Enterotoxigenic Escherichia coli (ETEC) produces different virulence factors allowing the bacterium to colonize and develop watery diarrhea. Proteomics studies have also introduced new protein belonging to the secretion pathways, antigen 43 (Ag43), which plays important role in E. coli pathogenesis. The objective of this study was to investigate O-types and virulence factors of E. coli isolates from neonatal calves diarrhea. Total of 120 isolates from diarrheic calves were genotyped for their O groups and the presence of virulence genes K99, F41 and STa as well as Ag43. The predominant O-type was O101 (51.00%) and the prevalence of K99, F41 and STa was 7 (5.80%). The Ag43 was detected in all samples with three different allelic patterns. Our results indicated that K99 positive isolates certainly have one of each 2200 bp or 1800 bp or both copies of Ag43 passenger domain, while negative K99 isolates lack the Ag43. The results reported here provide informative data regarding the prevalence of E. coli O-types and their virulence factors in enteric colibacillosis. The Ag43 that was more found in K99 positive isolates might be associated with diarrhea-causing E. coli strains in neonatal calves.