The directive calls for humane treatment but does not define it, leaving the issue to a separate directive that is still being debated, Pentagon spokesman Bryan Whitman said.
The directive sets a broad policy on which a new army field manual on interrogations and other rules will be based, he said.
It applies to all US military personnel, civilian defence contractors and other government agencies conducting interrogations of prisoners under US military control, according to the document.
Mr Whitman said it was the first time the Defence Department has issued such a directive on interrogation policy.
"All captured or detained personnel shall be treated humanely, and all intelligence interrogations, debriefings or tactical questioning to gain intelligence from captured or detained personnel shall be conducted humanely, in accordance with applicable law and policy," the directive states.
"Acts of physical and mental torture are prohibited," it said.
The law of war, relevant international law, US law, and other directives should be applied, it said.
They include a yet to be approved directive on detention policy that will define what is meant by humane treatment, Mr Whitman said.
"That particular document is still in final coordination. It will establish the requirements and the policies for holding, for processing, for transferring and releasing detainees, among other things," he said.
Debate within the administration has flared over whether humane treatment of prisoners should be defined with language taken directly from the Geneva Conventions.
The US has long refused to extend Geneva Convention protections to prisoners captured in the war on terrorism, while pledging to treat them humanely.
The torture debate gathered pace with last week's revelation by the Washington Post that since September 11, 2001 top al-Qaeda captives have been held in CIA-run prisons in at least eight countries.
The CIA, which is not part of the Defence Department, operates in secrecy, and the rules under which it works are not publicly known.
Vice President Dick Cheney has pressed Congress to exempt the CIA from legislation sponsored by Senator John McCain that would ban "cruel, inhuman and degrading treatment of prisoners in the detention of the US government".
But the White House said it had not asked Congress for a CIA waiver.