The plane blasted off from Cape Canaveral atop a SpaceX Falcon Heavy rocket which is capable of delivering it to a higher orbit than ever before.
The U.S. Space Force conducted the launch which came after more than two weeks of false starts and delays due to poor weather and technical issues.
It's the spaceplane's seventh mission since 2010.
The Pentagon has given few details about how high it will fly this time.
But looking at official statements, industry analysts and amateur space trackers speculate the X-37B may be bound for a highly elliptical orbit around EartH or even a path that could swing it out to the vicinity of the moon, a region of space in which the Pentagon has taken an increasing interest.
The spaceplane is also carrying a NASA experiment to study how plant seeds are affected by prolonged exposure to the harsh environment of radiation in space.
The ability to grow crops in space has major implications for keeping astronauts alive during future long-term missions to the moon and Mars.
Thursday's launch comes two weeks after China blasted its own equally secretive Shenlong robot spaceplane - on a launch system less powerful than SpaceX's rocket.
Still as the U.S.-China rivalry in space heats up, Space Force General B. Chance Saltzman had earlier this month said he expected China to launch Shenlong around the same time as the X-37B in what's seen as a competitive move.
X-37B's mission is expected to last until 2026.
