Background: Toxoplasma gondii is an important protozoan parasite infecting humans and almost all warm-blooded animals. As the only definitive host, cats play a crucial role in the transmission of T. gondii infection by shedding parasite oocysts in their feces. However, little information on T. gondii infection in cats was available in Lanzhou, northwest China. This study was performed to determine the seroprevalence of T. gondii infection in household and stray cats in Lanzhou, northwest China. Results: A total of 221 (179 households and 42 strays) blood samples were collected from clinically healthy cats admitted to several pet hospitals located in Lanzhou City, between November 2010 and July 2011 for the serological detection of T. gondii infection. The majority (207) of these cats represented Chinese Lihua cats. 47 of 221 (21.3%) examined cats were seropositive for T. gondii infection using the modified agglutination test (MAT) at the cut-off of 1:25. The seroprevalence in household and stray cats was assessed to be 15.6% and 45.2%, respectively, and the difference was statistically significant (P < 0.05). The seroprevalence ranged from 15.1% to 25.8% among different age groups, but the differences were not statistically significant (P > 0.05). Studies showed that there was no relationship between seroprevalence and the gender (P > 0.05). Conclusions: The present survey indicated the high seroprevalence of T. gondii in cats in Lanzhou, northwest China, which poses a threat to animal and human health. Therefore, measures should be taken to control and prevent toxoplasmosis of cats in this area.