This study was carried out to determine the antibiotic susceptibility profile and the production of metallo-β-lactamase (MBL) by Escherichia coli isolated from cow dung in Owo metropolis. The isolation of Escherichia coli was done using MacConkey agar and Eosin Methylene Blue Agar and was conventionally characterized. Antimicrobial susceptibility test of the isolates were by disc diffusion method against ceftazidime (30 µg), cefuroxime (30 µg), gentamicin (10 µg), cefixime (5 µg), ofloxacin (5 µg), augmentin (30 µg), nitrofurantoin (300 µg) and ciprofloxacin (5 µg). Detection of MBL producing isolates was by imipenem-EDTA combined disc test. The isolates showed highest resistance to augmentin (97.8%) and least resistance to nitrofurantoin (20.0%). Out of the 45 Escherichia coli isolated from cow dung, 8 (17.8%) produced MBL and were all multidrug resistant. The production of MBL and the high prevalence of antibiotic resistance observed among the Escherichia coli in this study infer that cow dung does not only serve as a reservoir for MBL-producers but also as source for the growth and dissemination of clinically significant antibiotic resistant species. Hence, the use of antibiotics as growth enhancers in cow production should be discouraged to help prevent the spread of antibiotic resistant bacteria and thus, preserve the efficacy of available antibiotics.