Avianca will suspend its daily service between Lima and Orlando from 1 July, as the airline continues to trim its network to improve profitability.
Its exit will leave LATAM Airlines Group as the only airline operating nonstop between the two cities, Cirium schedules data show.
Avianca says it will continue to serve Orlando from Bogota, and will boost frequency on the route to 11-times weekly from daily in July.
The Star Alliance carrier previously announced plans to cut Orlando service from Guatemala City, with effect from 1 June.
Avianca has dropped several routes in recent months as part of a wider strategic plan to improve profitability. In its US network, it ended service to both Boston and Chicago O'Hare in May.