The fighters from the Southern Movement took over six checkpoints in Ataq, the capital of Shabwa province, without facing any resistance, said the witnesses.
Policemen were told to give up their weapons and return to their bases, before the militiamen raised flags of the formerly independent South Yemen at the checkpoints.
The move comes after other southern cities and provinces declared they would disobey the central government in Sanaa.
The security forces have said they will defy any orders from the Yemeni capital after President Abdrabuh Mansur Hadi handed in his resignation on Thursday.
A committee in charge of military and security in four southern provinces, including the former South Yemen capital of Aden instructed troops and police to take orders from provincial governors and the fourth military region command in Aden.
Officers of the fourth military command are loyal to Hadi, himself a southerner.
Gunmen from the Popular Committees, which fought alongside government forces against Al-Qaeda in the past, have deployed in many areas, mainly Aden.
The committees were coordinating with the security committee and military regional command, he said.
"We have set up checkpoints to protect the city from gunmen, whether Huthis, or Qaeda militants," he said.
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