The gel, PRO 2000, is intended for use before sexual intercourse to help reduce HIV infection.
It was tested in a trial involving 9,385 women in four African countries: Uganda, South Africa, Tanzania and Zambia.
The risk of HIV infection was not significantly different among women supplied with the gel than in women given a placebo gel.
It was hoped microbicide gels would prove to be an effective way to limit the spread of HIV, as experts admit that condom promotion alone has not controlled the epidemic.
New ways of curbing the spread of HIV are badly needed, particularly in sub-Saharan Africa, where nearly 60% of those infected with the virus are women.
Women are often forced to take part in unsafe sex, and are biologically more vulnerable to HIV infection than men – so in theory a gel they could apply themselves could be effective.
A previous, smaller trial suggested PRO 2000 could reduce the risk of HIV infection by 30%.
But the latest study, carried out by the Microbicides Development Programme, a not-for-profit partnership of 16 African and European research institutions, failed to find any positive effect.
And the researchers say the trial was large enough to provide conclusive results.
The women who took part were given the gel together with free condoms and access to counselling about safe sex.
Important result
Lead researcher Dr Sheena McCormack, of the Medical Research Council, which part-funded the study, said: "This result is disheartening.
"Nevertheless, we know this is an important result and it shows clearly the need to undertake trials which are large enough to provide definitive evidence for whether or not a product works."
Professor Jonathan Weber, from Imperial College London, who also took part in the study, said: "It is unfortunate that this microbicide is ineffective at preventing HIV infection, but it's still vital for us as scientists to continue to look for new ways of preventing HIV.
"Now that we know this microbicide is not the answer, we can concentrate on other treatments that might be."
A similar research which taking place in Rwanda was halted.