The Gender Trust, of Horsham and Henfield, West Sussex, is awarded pounds 33,700 in grants to health, disability and care projects totalling pounds 9.6m.
The money for the charity, aimed at helping relieve emotional stress suffered by transsexuals and those uneasy about their sexuality, will go towards relocation and expansion.
The award was criticised by Tory MPs and, implicitly, by Virginia Bottomley, Secretary of State for National Heritage.
The largest grant is pounds 500,000 to the Samaritans to develop and fund their helpline.
The Child Migrants Trust, based in Nottingham, receives pounds 197,000 to expand its work of re-uniting children who were sent to live in Australian Children's Homes after the Second World War.
The National Association For Premenstrual Syndrome, in Sevenoaks, Kent, is awarded pounds 161,112, which will be used to raise awareness of the condition.
Other groups receiving awards included the Leonard Cheshire Foundation for the disabled (pounds 492,203), the Burma Star Association for war veterans (pounds 83,454), the Royal National Institute for the Blind (331,714), Cancer Relief Macmillan Fund (pounds 150,000) and Community Service for Volunteers (pounds 180,000).
Harry Greenway, Tory MP for Ealing North, said: "Support for transsexuals is not the right use of what amounts to public money."
Mrs Bottomley said: "I am delighted that the National Lottery Charities Board have decided to support medical charities and others including the Samaritans, the RNIB and the Burma Star Association which will be widely welcomed.
"However, it is for the Charities Board to explain and account for their actions in the areas which may not attract widespread public endorsement."
In the past John Major has expressed opposition to donations of lottery cash to organisations for groups such as lesbians. Last month the NLBC awarded pounds 75,000 to an advice group for male prostitutes.