Manchester's small theatre, situated in two converted Victorian railway arches beneath Oxford Road railway station, has announced it will close at the end of May.
In a statement, Greenroom said: "Following the recent disappointing news that Greenroom is not part of Arts Council England's National Portfolio for 2012/13 onwards, the Board has made a decision that Greenroom, Manchester, will close to the public at the end of May this year.
"We would like to have continued but are unable to do this without confirmed funding or alternative income going forward.
"Greenroom, since its inception, has been at the forefront of discovering and pioneering Manchester, national and international 'firsts'. We have been consistently committed to developing and presenting new and experimental performance, and more recently supporting and enabling artists at the start of their careers to be innovative, take risks and experiment with new ideas.
"Our aim is to ensure that this important work continues through other organisations and individual artists/artist companies, producers and promoters.
"We are working with the Arts Council to provide information for people interested in the future production of this work and this will be available shortly on request by emailing firstname.lastname@example.org
"It is with regret that we find ourselves in this situation almost 25 years since the venue opened , and would like to extend our thanks to all of you who have recently sent us words of support.
"We would like to thank our long-standing funders: Arts Council England, AGMA and Manchester City Council, who have shown a significant commitment in supporting Greenroom.
"The current board and staff feel proud and privileged to have been part of Greenroom; and we'd like to thank everyone – including artists, audiences and partners – who have contributed to its rich history, and particularly to those who have at some point called Greenroom 'home'."
Facilities at the Greenroom include a 200-seat theatre, a studio style rehearsal room, exhibition facilities, a cafe/bar and offices.
Arts Council England is having its budget reduced by £100m by the Government from 2012-2015.
ACE received 1,333 applications to join its new National portfolio and said it has offered funding to 695 organisations. ACE used to regularly fund 849 organisations.
ACE said 110 new organisations are being brought into the mix, with decisions shaped by a ten year vision for the arts.
ACE's National portfolio funding is being supported with £18m Lottery funding a year for earmarked portfolio organisations.
A total of £10.5m Lottery funding a year is also targeted at work with children and young people, to ensure educational work continues, while an extra £12m of grants for the arts Lottery money is being freed up for small organisations.