BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is the main causative agents of neonatal calf diarrhea. Passive transfer of antibodies that is derived from the dams of the calves through their colostrum plays an important role in disease prevention. OBJECTIVES: To determine the presence of natural specific antibodies against K99 antigen in dam’s serum and colostrum as well as in calf’s serum and examined the association between bacteriological and serological aspects for ETEC K99+ and calf diarrhea. METHODS: A specific PCR assay was used to detect K99-positive E. coli in fecal samples from two groups of normal and diarrheic calves. Specific antibodies against K99 fimbriae in dam serum, colostrum and calf serum were studied using indirect ELISA. RESULTS: Significant differences were observed between the antibody titer in normal and diarrheic groups. Significant differences were observed between normal and diarrheic calves in their serum anti-K99 titers (P =0.0005), their dams (P =0.0005) and colostrum (P =0.001), in which anti-K99 titer in dam’s serum and colostrum was higher in diarrheic group, but in calf’s serum was higher in normal group. CONCLUSIONS: Although anti-K99 antibody levels in diarrheic calves was less than normal calves, there is no correlation between the level of these antibodies and disease occurrence. This study suggests that anti-K99 antibodies do not solely promote immunity to ETEC infections.