The latest resignation came a day after prosecutors launched a probe into allegations that police officials ran a male prostitution network from 2004 to 2008.
Police chief Rodolfo Palomino, 58, who is accused of creating the ring, made a public appearance with his wife and children in which he said he was resigning "in the absolute and full certainty that I am not guilty of any of the charges against me."
"Faced with the known facts... I have made the decision with my family and with the police generals to ask the president to remove me from my post," he said, drawing applause from supporters.
Prosecutors said in a statement Tuesday they were investigating Palomino over the "creation and running of an alleged male prostitution network known in the media as 'The Fellowship of the Ring.'"
A video broadcast by Colombian media supposedly shows deputy interior minister Carlos Ferro in 2008, when he was a senator, discussing male prostitutes.
Ferro, 53, resigned on Tuesday over that video, sources close to the case told AFP.
The state prosecution service is headed by Alejandro Ordonez, a conservative political opponent of President Juan Manuel Santos.
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