The ministry said in a Facebook post that the chopper went missing in the rugged Cardamom Mountains, which are cloaked in dense rainforest.
"A helicopter has lost contact with the headquarters of the air force... during a training," it said.
"The accident happened due to bad weather," it added.
The ministry did not say when the chopper went missing, what model it was, or how many people were on board.
But air force sources told AFP that a Chinese-made Z-9 chopper with at least two people aboard disappeared Friday during a training exercise.
The defence ministry said rescue teams began searching for the missing Saturday morning, but the chopper had not been found.
Cambodia bought 12 Z-9 helicopters from China in 2013 to boost its military capacity.
Four Cambodian soldiers died and one was injured in 2014 when a Z-9 exploded in mid-air before crashing into a water-filled quarry during military training on the outskirts of the capital Phnom Penh.
In 2008, Cambodia's chief policeman Hok Lundy was killed in a helicopter crash along with the then-deputy army commander and two pilots when their chopper went down in bad weather.
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