No reason was given for Pyongyang's decision to rescind Ambassador Robert King's invitation. (Agencies)
But State Department spokeswoman Jen Psaki stressed that North Korea had promised last year it would not use jailed Christian missionary Kenneth Bae as a "political bargaining chip."
Bae, a US citizen and tour operator described by a North Korean court as a militant Christian evangelist, was arrested in November 2012 and later sentenced to 15 years' hard labor on charges of seeking to topple the government.
Bae, who is also known as Pae Jun-Ho, was detained as he entered North Korea's northeastern port city of Rason.
US officials say Pyongyang has moved him from hospital back to a labor camp, saying they were "deeply concerned" for the health of the 45-year-old Korean American, who has been detained for more than 15 months.
"We are deeply disappointed by the DPRK decision -- for a second time -- to rescind its invitation for Ambassador King to travel to Pyongyang to discuss Kenneth Bae's release," Psaki said, using North Korea's formal acronym.
"We again call on the DPRK to grant Bae special amnesty and immediate release as a humanitarian gesture so he may reunite with his family and seek medical care. We will continue to work actively to secure Mr. Bae's release."
South Korea and the US meanwhile announced they would begin joint military drills on February 24, but Psaki insisted that this was not intended to pressure Pyongyang to release Bae.
"We remind the DPRK that the US-ROK military exercises are transparent, regularly-scheduled and defense-oriented," she said.
"These exercises are in no way linked to Mr Bae's case," she said. Washington and Pyongyang have no diplomatic ties.
No reason was given for Pyongyang's decision to rescind Ambassador Robert King's invitation.