Air travel between the United States and Cuba had been restricted to charter flights from 1979 until earlier this year
The departure of an American Airlines flight from Miami at 7:30 am (1230 GMT) came just days after the death of former Cuban leader Fidel Castro at age 90, as throngs of Cubans flock to Havana's Revolution Square to pay last respects to the divisive Cold War titan.
"It's a once in a lifetime experience, it will be interesting to see how people are responding to his passing," passenger Priva Rhat told reporters on the flight, which set off from Miami with a water cannon salute.
In New York, the first JetBlue Airways flight bound for Havana departed an hour and a half later.
Air travel between the United States and Cuba had been restricted to charter flights from 1979 until earlier this year.
Direct commercial flights began on August 31, linking several US airports with nine Cuban cities, many of them in or near tourism hotspots.
There are now 110 daily flights from the United States to Cuba, 20 of them to Havana.
The direct flights were 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.
Diplomatic relations were restored in July 2015.
Washington still bans Americans from visiting Cuba as tourists, but travel is permitted for 12 other categories, including cultural and educational exchange.