Miyazaki prefectural officials culled all the chickens at a farm in Nobeoka city, the country's top poultry producing region, in an operation that started at 2:30 am (1730 GMT Monday), the Kyodo Newsagency reported.
The local government told nearby poultry farms to restrict the movements of livestock.
Sixteen birds died at the farm on Monday, of which three tested positive for bird flu in preliminary tests, the report said.
The case was the country's first since April, when 110,000 chickens were culled in neighbouring Kumamoto prefecture after a bird flu case was detected there.
In 2011, about 1 million chickens were culled in Miyazaki prefecture as H5 avian influenza spread in the region.