Riding on Nasir Jamshed's second consecutive century, the visitors posted 250 all out and then shot out the Indians for a paltry 165 to record a convincing victory with two overs to spare at the Eden Gardens.
It was yet another woeful display by the famed Indian batting line-up, which has been struggling for sometime now and has been largely responsible for the team's forgettable results in recent times.
The huge defeat to the arch-rivals will now raise questions on the future of some of India's under-performing batting stars, who have let the team down more often than not.
Jamshed (106) notched up his third ODI century, all three coming against India, but Pakistan suffered a dramatic collapse after being at a healthy 141 for no loss at the mid-way stage. Mohammed Hafeez was the other contributor with a rollicking 76 off 74 balls.
The floodgates opened up after the dismissal of Hafeez as none of the other batsmen could hang around for long on a slow Eden Garden track, which made stroke-making difficult after the ball became older.
The Indians needed a good start from their struggling openers -- Gautam Gambhir and Virender Sehwag -- but it did not materialise yet again, putting pressure on the jittery middle-order.
Sehwag (31), Gambhir (11), Virat Kohli (6), Yuvraj Singh (9) and Suresh Raina (18) perished in quick succession to give the visitors the upper hand in what eventually turned out to be a lop-sided contest.
Skipper Dhoni, who scored a heroic unbeaten 113 in the first ODI in Chennai, again provided some resistance with a cautious and unbeaten 54 but wickets kept tumbling at the other end to nullify his efforts.
The series defeat against Pakistan marks another low for the struggling team which suffered an embarrassing Test series debacle against England at home and levelled a T20 series 1-1.
Thursday's defeat has also rendered the third and final ODI at Ferozshah Kotla in Delhi on Sunday inconsequential.
After giving the first breakthrough, Jadeja took two wickets -- Jamshed and Kamran Akmal -- in one over to trigger a collapse and Ishant Sharma (3/34) polished off the tail.
From 141 for no loss, Pakistan lost 10 wickets in 25 overs as India made a spectacular comeback.
At the start, the inexperienced Indian attack of Ashok Dinda and Bhuvneshwar Kumar could not make use of the overcast conditions.
In his 'home' debut, Dinda was wayward with his bouncers, conceding three wides -- two in his second over -- while Bhuvneshwar too lacked the bite as the Pakistani duo went scotfree.
The 23-year-old Jamshed continued from where he had left in Chennai, while Hafeez made up for his duck with a sensible batting display and rotating the strike.
The duo never panicked and such clinical was their approach that they put on 100 runs in exactly 100 balls --incidentally, Pakistan's first century stand by any opening pair at the Eden.
Part-time spinner Jadeja finally gave the breakthrough almost at the midway stage when Hafeez missed the ball trying to break free.
Azhar Ali (2) continued his poor run when he was run out after being sent back by Jamshed as Pakistan lost their second wicket in four runs.
They quickly lost third wicket as Younis Khan (10) was adjudged leg before despite a bat-pad. Jamshed, however, kept his cool to bring up his second consecutive century.
Jamshed clobbered Jadeja over his head for a straight boundary to move to 96. He straight drove one uppishly in the mid-off boundary region to bring up his hundred.
But he could not carry his innings through as he became Jadeja's second victim. Jadeja claimed Kamran Akmal (0), his third, in the same over to trigger Pakistani middle order collapse.
India lost to Pakistan last time around at home in 2004-05 when they lost the six-match series 2-4.
And now India have lost all the four matches they have played against Pakistan at Eden Gardens.
Chasing a modest 251, the under-fire opening duo of Gambhir and Sehwag had a sedate start. Sehwag scored a few boundaries but just when the partnership looked like building, Junaid provided the breakthrough in the form of Gambhir.
The left-hander chased a widish delivery and the ball hit his stump after taking an inside edge of his blade.
It was a familiar story from there on as the failing middle-order found the going tough against some disciplined fast bowling of Umar Gul and Junaid.
India were reduced to 70 for four as promising Kohli continued and Yuvraj could not get to double digit score, both being dismissed caught behind taking thin edges.
As India stuttered, Gul almost dismissed Raina running into the non-striking end but his throw missed the stumps with the Indian left-hander diving forward.
Raina also started his walk back when he was stumped off Hafeez.
Promoted ahead of Jadeja, Ashwin (3) did not last long going down to the guile of Shoaib Malik for yet another stumping.
Jadeja (13) could not give the support Dhoni needed in the middle when he was caught at mid-on as it was just a matter of time for the visitors to seal the series.
Earlier, under overcast conditions, Jamshed scored a fluent 106 from 126 balls and along with Hafeez put up a handsome 141-run opening partnership.
But India made a dramatic comeback riding on left-arm spinner Ravindra Jadeja's 3 for 41 as Pakistan were bundled out in 48.3 overs.