Objectives Trichomonas vaginalis is thought to be the most common non-viral sexually transmitted infection worldwide. We investigated the prevalence, risk factors and protozoan load of T. vaginalis infection in South African women. Methods A cross-sectional study of 604 women was conducted at 25 primary healthcare facilities in rural South Africa (Mopani district). T. vaginalis DNA was detected in vaginal and rectal swabs. In univariate and multivariate analyses, the T. vaginalis infection was investigated in relation to demographic characteristics, medical history and behavioural factors. The T. vaginalis load was determined as the logarithm of DNA copies per microlitre sample solution. Results Collected vaginal and rectal swabs were tested for T. vaginalis DNA. Prevalence of vaginal T. vaginalis was 20% (95% CI 17.0% to 23.4%) and rectal 1.2% (95% CI 0.6% to 2.4%). Most women (66%) with a vaginal infection were asymptomatic. Factors associated with T. vaginalis infection were a relationship status of single (OR 2.4; 95% CI 1.5 to 4.0; p<0.001) and HIV positive infection (OR 1.6; 95% CI 1.0 to 2.6; p=0.041). Women with vaginal T. vaginalis infection were more likely to have concurrent Chlamydia trachomatis rectal infection than those without vaginal infection (12%vs3%; p<0.001; OR 4.1). A higher median T. vaginalis load was observed among women with observed vaginal discharge compared with those without vaginal discharge (p=0.025). Conclusions Vaginal trichomoniasis is highly prevalent in rural South Africa, especially among single women and those with HIV infection, and often presents without symptoms.