An SFL spokesman said: "I have this morning received intimation from the Gretna FC administrator of their resignation from the Scottish Football League with immediate effect.
"I have accepted this resignation on behalf of the Scottish Football League and accordingly, we now have a vacancy in the Third Division and are seeking applications."
Gretna's resignation promises a busy summer for the SFL and potential applicants for the vacancy in the Irn-Bru Third Division.
The SFL have set a deadline of Thursday June 12 for prospective new clubs to apply for membership.
The SFL Management Committee will then visit each applicant club by Friday, June 27.
Applicant clubs will then be invited, following assessment, to present their business and football plans to all 29 members of the SFL at a Special General Meeting to be held at Hampden Park on Thursday, July 3.
The 29 SFL clubs will then vote on the day to decide which club has been invited to join the SFL as a Third Division club with effect from the 2008-09 season.