The 34-year-old Kenyan completed the course in Vienna, Austria, in an incredible one hour, 59 minutes and 40 seconds.
Kipchoge was racing alone but assisted by 41 pacemakers, including former Olympic and world record holders, in his attempt to achieve the milestone.
The time will not be an official world record but is nonetheless a historic moment in distance running.
The INEOS 1:59 Challenge was Kipchoge’s second shot at the record after missing out by 25 seconds in his first attempt in Monza two years ago.
Kipchoge was even able to wave to the crowds as he sprinted towards the finish line, where his wife Grace greeted him.
In jubilant scenes Kipchoge was somehow able to keep running as he celebrated with the crowd before being hugged by his INEOS team-mates.
Afterwards he told the BBC: “I am feeling good. After Roger Bannister in 1954 it took another 63 years, I tried and I did not get it.
“After 65 years, I am the first man! I want to inspire many people, that no human is limited.”
Kipchoge tore up the specially tailored course over 4.4 laps of the Prater Hauptallee, a park in central Vienna.
Guided by lasers on the road from a support car in front and surrounded by seven pacemakers at a time, the world record holder stuck to a pace of 2min 50sec per kilometre.
He hit the hour-mark with 11 seconds to spare and with 500m to go burst clear of his support team to finally break the hallowed two-hour barrier.
Kipchoge, the four-time London Marathon winner whose official world record time is 2:01.39, added: “Remember the 41 pacemakers are among the best athletes ever in the world.
“I can say thank you to them, I appreciate them for accepting and together we made history on this one.
“We can make this world a beautiful world and a peaceful world. My wife and three children, I am happy for them to come and witness history.
“The positively of sport, I want to make it a clean sport and an interesting sport.”