We estimated the prevalence of HIV and assessed correlates of HIV infection in long-distance truck drivers in South Africa. Between October 2003 and July 2004, 1900 long-distance truck drivers aged ≥18 years consented to interview and for testing for HIV. Participants were selected from a 10% stratified random sample of registered truck depots. A proximate-determinants framework was used to assess the hierarchical relationship between risk factors and HIV infection using logistic regression. HIV prevalence was 26% (95% confidence interval 24% to 28%). In multivariate analyses, HIV infection was associated with spending 2–4 weeks on the road (adjusted odds ratio 1.4; 95% confidence interval 1.1 to 1.9). There was modest evidence of a dose-response relationship between time on the road and HIV risk. Mobility increased risk by creating conditions for unsafe sex and reducing access to health services. Targeted HIV interventions for long-distance truck drivers are needed.