Background: Cryptosporidium is a protozoan parasite causes cryptosporidial diarrhea, which is typically a short-lasting benign infection, but can become severe and non-resolving in immunocompromised individuals. The aim of the present study was to de-terminate the prevalence of Cryptosporidium infection in livestock and humans that were in contact with livestock in Hamadan district, Iran.Methods: In this cross sectional study a total of 660 fecal specimens were collected; 228, 195 and 237 from humans, whom raising livestock, their calves and lambs/goats, respectively in spring 2012. Samples were concentrated by formalin-ether concentration technique and examined using cold modified Ziehl-Neelsen staining method.Results: Two (0.87%) out of 228, 25 (12.8%) out of 195 and 6 (2.5%) out of 237 fecal samples of humans, calves and lambs/goats were positive for Cryptosporidium oocysts, respectively. There was no significant relationship between the infection to Cryptosporid-ium and demographic variables of humans. However, Cryptosporidium infection rate was higher in diarrheic calves (OR=3.81; 95% CI: 1.30, 11.21; P=0.010).Conclusion: Despite studies conducted in some regions of Iran that resulted in a rela-tively high rate of infection in humans in contact with livestock, our results showed low prevalence and low carrier status in the asymptomatic persons in Hamadan region. Be-cause the infection in calves and lambs/goats was relatively high, these animals could be probable reservoir of infection for humans in this area.