Ward, the world number 111, recovered from two sets down to win 6-7 (5-7) 5-7 6-3 7-6 (7-3) 15-13 in Glasgow.
The Englishman needed six match points before finally seeing off the world number 20 after almost five hours.
Andy Murray, playing his first match in Scotland for four years, had earlier beaten Donald Young 6-1 6-1 4-6 6-2.
Media playback is not supported on this device
The best-of-five tie continues with Saturday's doubles, before the reverse singles on Sunday.
"It's really tough for the USA now at two matches down," said GB captain Leon Smith.
"But they have Jim Courier as captain and he will not let them go down without a fight."
There is now every prospect of Britain repeating last year's win over the US at the same stage, after Ward again produced an upset in the second singles match.
Last year it was Sam Querrey in four sets - this time the Londoner came through a 111-minute final set to see off Isner and claim the win of his life.
And if Murray had been expected to dominate proceedings on his return home, the opening day ended with Ward acclaimed by the 7,700 spectators at the Emirates Arena.
"This is by far the best atmosphere I've ever been involved in and thank you very much to all the crowd," said Ward.
"It was difficult. I had a lot of chances in the last set. It was an unbelievable match."
A downcast Isner admitted: "It's brutal. I'm barely going to sleep tonight, it's awful.
"I'm healthy. Mentally I'm certainly a bit shaken right now but I've got to be a professional and bring my best on Sunday. I don't feel too chipper right now but I'll let it rip on Sunday if that match is live."
Isner looked in control after taking the opening tie-break and breaking serve for the first time in the match on his way to a two-set lead.
Ward has been in the best form of his career over the last 12 months, however, and backed up by a gruelling off-season training block with Murray, he dug in and worked his way back into the contest.
Two terrific passes helped him finally break the Isner serve in the third set and the Briton levelled in a tie-break as the momentum swung steadily in his favour.
Ward dominated the decider, earning a break point and then five match points, only for the 6ft 10in Isner to keep him at bay with his huge serve.
With no tie-break in the deciding set, the games ticked by, prompting memories of Isner's 11-hour marathon against Nicolas Mahut at Wimbledon 2010.
Ward had his own moment of danger at 11-11 but saved two break points, the second in a long and desperately tense rally, but for the most part he looked the more likely winner.
Media playback is not supported on this device
It took until the 28th game of a gripping final set but there was no escape when Isner slipped 0-40 down, and he netted a volley to spark wild celebrations among the British squad.
Ward shared a long hug with Murray, who had been dispensing advice via Smith from the sidelines throughout.
The Scot had earlier seen off Young with a fine display in front of his home crowd, who gave him a rousing reception.
"It was very nice," said the Scot. "I used the energy and emotion very well at the beginning of the match. I would say I played extremely well.
"I'm going to praise myself today - I hit one unforced error in two sets."
James Ward produced a stunning comeback to beat John Isner 15-13 in the fifth set and give Great Britain a 2-0 Davis Cup lead over the United States.