Scheduled air service between the US and Cuban international airports other than Havana's Jose Marti International Airport would be barred in 45 days, the State Department said in a statement on Friday.
This move aims to prevent the Cuban government from profiting from US air travel, the statement added.
Cuban Foreign Minister Bruno Rodriguez condemned the move on Twitter, saying it "strengthens US citizens' travel ban; mutilates US people liberties & damages people-to-people contacts"
"But they won't extract any concession from us. We shall overcome!" Rodriguez wrote.
Friday's move comes after the US last week announced a series of fresh sanctions against Cuba in retaliation over Havana's support for Venezuelan President Nicolas Maduro.
The new sanctions came into effect on October 21.
Since taking office in 2017, US President Donald Trump has toughened Washington's diplomatic and economic stranglehold on Cuba by reducing diplomatic staff, imposing new trade sanctions, banning Cuban vessels and restricting American citizens' travel to the island.
These aggressive measures have had noticeable effects on Cuba's economy, particularly in areas such as tourism, which has seen a marked decline in the number of visitors.
Cuba was taken off the list in 2015 during the administration of Trump's predecessor, Barack Obama, who sought to reduce diplomatic tensions with Havana during his two terms in office.
( With inputs from IANS )