Libratus, an artificial intelligence programme developed at Carnegie Mellon University, was trained to play a variant of the game known as no-limit heads-up Texas hold 'em.
In a similar tournament in 2015, the humans won.
The victory has been hailed as a significant milestone for AI, by the team responsible for building it.
The AI won more than $1,500,000 worth of chips from the humans.
The matches - held at Rivers Casino in Pittsburgh - were live-streamed over gaming site Twitch.
Tuomas Sandholm, professor of computer science at Carnegie Mellon, said the event was 'historic'.
"Heads-up no limit Texas hold 'em is in a way the last frontier of all the games," he said.
"Othello, Chess, Go, Jeopardy have all been conquered, but this remained elusive: this is a landmark in AI game-play."
Prof Sandholm said that the algorithm could be transferred to a range of other uses.
"This is not just about poker," he said.
"The algorithms can take information and output a strategy in a range of scenarios, including negotiations, finance, medical treatment and cybersecurity."
"Now we have proven the ability of AI to do strategy and reasoning, there are many potential applications in future."


