A seroepidemiological study was performed to determine the seroprevalence of Cryptosporidium in human immunodeficiency virus (HIV)-infected adults and local university students in the Limpopo Province, South Africa. Using a custom anti-C. parvum immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), the seroprevalence of Cryptosporidium was found to be significantly higher (75.3%; 146 of 193) in HIV-infected individuals compared with student volunteers (32.8%; 19 of 58) (P < 0.001). A more recent diagnosis of HIV was associated with anti-C. parvum IgG seropositivity, as was lower weight among HIV-infected women. This is the first seroepidemiologic study of Cryptosporidium in rural South Africa, and it shows high endemicity among the HIV-infected population. In addition to raising the possibility of significant Cryptosporidium-related morbidities, this finding reveals that in Limpopo and perhaps in other low-income, rural populations, interrupting waterborne pathogen transmission will require strategies effective against environmentally hardy parasites such as Cryptosporidium.