An MC-130E Combat Talon I aircraft dropped the bomb at 2:30pm (9:30 GMT) on a test range in Florida, sending a plume more than 3300 metres above the ground.
"The bomb separated cleanly from the aircraft at 7000 metres and with its Global Positioning System and navigation system glided to its target area on Eglin's test range," the air force said.
The 10 metres long, 102cm wide bomb is officially named the Massive Ordnance Air Blast bomb, but is more popularly known as the Mother Of All Bombs.
Developed in nine weeks and pressed into service in Iraq after an initial test on 11 March, the Moab contains 8500kg of high explosives.
Shipped to Gulf
Several of the weapons were shipped to the Gulf in April.
The Moab is a lot similar to the 6800kg "daisy cutter", which was used to raze jungle for helicopter landing pads in Vietnam, to clear minefields in the 1991 Gulf War and more recently to blow out caves in Afghanistan.
Lynda Rutledge, manager of the Moab programme, said the latest test was designed to prove its effectiveness.
"There were minor modifications to the weapon dropped today compared with the earlier one," Rutledge said. "What we were trying to do was give our combatant commanders an opportunity to understand all its implications and understand how it performs.
The Moab has a satellite guidance system and a tail kit to steer it to within about 13 metres of its target.
It is so big it has to be dragged out of the back of a C-130 cargo plane by a parachute.