Neospora caninum is an obligate intracellular parasite that can infect domestic and wild canids, as well as ruminants and equines. It was described in 1988 and has been known as a major cause of abortion in bovines and neuromuscular alterations and death in dogs. To estimate the prevalence of bovine neosporosis in the 22 municipalities of the so-called Estrato 1 subregion of the Brazilian state of Mato Grosso do Sul, blood samples were collected from cows aged 24 months and older, from December 2003 to March 2004. During sample collection, a questionnaire was used to gather data of epidemiological interest. The samples were subjected to serological diagnosis (indirect fluorescence antibody test - IFAT). Prevalences of 14.9% (449/2488) and 69.8% (143/205) were found for the animals and herds sampled, respectively. The variable found to be associated with seropositivity to N. caninum was abortion (OR 2.52; CI 1.25-5.06). The results revealed the presence of infection by N. caninum in the herds investigated, drawing attention to its role as a potential cause of abortion in cattle in Mato Grosso do Sul.