Shiga toxin-producing Escherichia coli (STEC) strains are commensal bacteria in cattle and cause food borne disease in human. We analyzed the isolation rate of STEC in stool specimens of patients with diarrhea and in fecal samples of cattle in Gwangju, Korea. STEC strains were detected from 33 (0.19%) out of 17,148 patients with diarrhea while there has been a progressive increase in the incidence rate from 0.07% in 2004 to 0.33% in 2008. We investigated serotypes, shiga toxin genes, and antimicrobial resistance patterns of the 44 STEC isolates from human and cattle sources. The 33 STEC isolates from human belonged to 14 O serotypes including O157, O26 and O111. The 11 isolates from cattle belonged to 11 O serotypes. PCR detection for stx genes showed that 12 (27.3%) isolates carried stx1 genes, 20 (45.5%) possessed stx2 genes, and 12 (27.3%) carried both stx1 and stx2. Of the 33 STEC isolates from human, 25 strains (76%) were resistant to one or more antibiotics. High level of resistance to tetracycline (73%) was most common, followed by ticarcillin and ampicillin (64%). But none of the 33 isolates from human were resistant to amikacin, cefazolin, cefepime, cefotetan, cefotaxime, ciprofloxacin, or imipenem. The 5 strains (45%) of the 11 isolates from cattle were resistant to at least one or three antibiotics but most of the isolates were sensitive to the 16 antibiotics employed in this survey. In conclusion, toxin types and serotypes of STEC isolated from human and cattle were diverse, and non-O157 STEC was also observed to be a greater proportion of STEC isolates. According to a specific comparison solely based on the toxin types and serotypes, most of the STEC strains isolated from cattle feces in Gwangju, Korea showed characteristics different from those isolated from patients. Therefore, laboratory surveillance is required to detect and carefully monitor the potentially hypervirulent STEC not only in human and cattle but also in other animals.