In a remark to a few people at the Massachusetts Institute of Technology last week, Crowley said the treatment of the accused WikiLeaks source in the Quantico, Va., brig is "ridiculous and counterproductive and stupid."
After BBC blogger Philippa Thomas reported the statement, President Barack Obama asserted at a Friday news conference Manning's conditions of detention were "appropriate and are meeting our basic standards."
Secretary of State Hillary Clinton said Sunday she accepted Crowley's resignation with regret, The New York Times reported. The resignation was first reported by CNN.
"P.J. has served our nation with distinction for more than three decades, in uniform and as a civilian," Clinton wrote in a statement.
Manning's lawyer says the soldier is being forced to be naked on the pretext of suicide prevention.
Crowley said he takes "full responsibility" for his comment.
"The exercise of power in today's challenging times and relentless media environment must be prudent and consistent with our laws and values," he added.