Toxoplasma gondii is widely distributed in humans and other animals, including wild rats throughout the world, but little is known of the prevalence of T. gondii in rats in China. The seroprevalence of T. gondii in rats (Rattus norvegicus and Rattus flavipectus) was investigated in Guangzhou, southern China, between November 2009 and January 2010. In total, 217 rat serum samples were collected; antibodies to T. gondii were detected by the modified agglutination test (MAT), and 7 (3.2%) were found positive (titers ≥1:40). The seroprevalence was higher (3.4%) in R. norvegicus than in R. flavipectus (3.0%), but the difference was not statistically significant (P > 0.05). All 7 positive rats were female; no T. gondii antibodies were detected in males. This is the first extensive survey of T. gondii infection in rats in southern China, and the results have public health implications in this region.