Few detailed epidemiologic data exist regarding the impact of HIV infection on the workplace in the developing world. In addition, most HIV surveys examine only prevalence, without data on incidence or disease severity. In June 2003, we conducted a voluntary anonymous HIV serosurvey among employees of the Debswana Mining Company, the largest nongovernmental employer in Botswana. Among the 3558 participants, annual HIV incidence was estimated to be 3.4%, and HIV prevalence was 23.8%. HIV-infected participants had a median CD4+ lymphocyte count of 427 cells/mm3 (interquartile range 269–642), with 13.3% of samples <200 cells/mm3. The high incidence and prevalence of HIV, and the proportion of workers with <200 CD4+ lymphocytes/mm3, demonstrate the impact of HIV infection in this workplace and the potential negative impact on productivity. These results highlight the need for routine HIV testing and access to antiretroviral treatment for workers in countries such as Botswana.