Toxoplasma gondii and Neospora caninum are closely related protozoan parasites which cause lowered production and increased abortion in dairy cows. The aim of the present study was to determine the seroprevalence of T. gondii and N. caninum infection in dairy cows in the Guangxi Zhuang Autonomous Region (GZAR), subtropical southern China. In total, 875 serum samples were collected from the tail veins of dairy cows in 6 main dairy cow-rearing districts of 4 administrative cities in GZAR. The samples were surveyed for T. gondii antibody using the Indirect Haemagglutination Test (IHA), and 365 of the serum samples were examined for N. caninum antibody by indirect Enzyme-Linked Immunosorbent Assay (ELISA). The overall seroprevalence of T. gondii in dairy cows was 13·71% (120/875), and the average seroprevalence of N. caninum was 15·07% (55/365). There were significant differences in the seroprevalence of N. caninum infection between different districts (P = 0·002, χ2 = 9·261). The highest prevalences of T. gondii and N. caninum were found in cows older than 8 years and those that had completed 5–6 pregnancies. Five cows (1·37%) presented antibodies against both T. gondii and N. caninum, and dairy cows with both T. gondii and N. caninum antibodies had higher abortion rates. The present results indicate widespread exposure of dairy cows to T. gondii and N. caninum in GZAR, subtropical southern China.