OBJECTIVES: To examine the impact of a large-scale HIV prevention programme for female sex workers (FSW) in Karnataka state, south India, on the prevalence of HIV/sexually transmitted infections (STI), condom use and programme coverage. METHODS: Baseline and follow-up integrated biological and behavioural surveys were conducted on random samples of FSW in five districts in Karnataka between 2004 and 2009. RESULTS: 4712 FSW participated in the study (baseline 2312; follow-up 2400), with follow-up surveys conducted 28-37 months after baseline. By follow-up, over 85% of FSW reported contact by a peer educator and having visited a project STI clinic. Compared with baseline, there were reductions in the prevalence of HIV (19.6% vs 16.4%, adjusted odds ratio (AOR) 0.81, 95% CI 0.67 to 0.99, p=0.04); high-titre syphilis (5.9% vs 3.4%, AOR 0.53, 95% CI 0.37 to 0.77, p=0.001); and chlamydia and/or gonorrhoea (8.9% vs 7.0%, AOR 0.72, 95% CI 0.54 to 0.94, p=0.02). Reported condom use at last sex increased significantly for repeat clients (66.1% vs 84.1%, AOR 1.98, 95% CI 1.58 to 2.48, p<0.001) and marginally for occasional clients (82.9% vs 88.0%, AOR 1.22, 95% CI 0.89 to 1.66, p=0.2), but remained stable for regular partners (32%). Compared with street and home-based FSW, brothel-based FSW were at highest risk of HIV and STI, despite high levels of reported condom use. CONCLUSIONS: This large-scale HIV prevention programme for FSW achieved reductions in HIV and STI prevalence, high rates of condom use with clients and high rates of programme coverage. Improved strategies to increase condom use with regular partners and reduce the vulnerability of brothel-based FSW to HIV are required.