JetBlue Flight 386 left Fort Lauderdale in southeastern Florida shortly after 10:00 AM (1400 GMT) with 150 passengers on board. After less than an hour in the air, it will land in central Cuba's Santa Clara, 280 kilometers east of Havana.
The flight is the first of dozens of daily trips connecting US cities to nine Cuban airports, many of them in or near tourism hotspots. Regular air service was severed during the Cold War, and charter flights have been the only air links since.
The Fort Lauderdale airport was in full party mode near Jet Blue's departure area -- a live salsa band blared Cuban favorites as passengers and bystanders broke into spontaneous dances.
There were cheers, applause and a sea of balloons as boarding for the historic flight got underway.
For some, there were also tears of joy.
"I am so proud, so overcome with emotion," said Domingo Santana, 53, who left Cuba when he was just six years old.
Today's JetBlue flight will be flown by Captain Mark Luaces and First Officer Francisco Barreras, both Americans of Cuban descent, the airline said.
The plane was sent off with a water cannon salute, an aviation tradition in which aircraft pass under arcs of water before flying to their destinations for the first time.
Mark Gale, director of operations for the airport, said it was one of the "great moments in history," likening it to the moon landing or the fall of the Berlin Wall.
"This reopening has really benefitted us," she gushed.
"Not only in terms of the ticket prices, but also in terms of how much easier it is to purchase them," she said.
"Now we only need more vacation time!"
The last regular commercial flight between the two countries took place in 1961, when air links fell victim to the Cold War.
Air travel between the United States and Cuba has been restricted to charter flights since 1979.
Washington and Havana agreed in February to restore direct commercial flights -- one of several watershed changes initiated in December 2014, when US President Barack Obama and Cuba's Raul Castro announced a thaw after more than 50 years of hostility.
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