Efficacy of an Escherichia coli (O111:B4) J5 bacterin was tested in an experimental challenge trial. Nineteen cows were vaccinated with an E. coli J5 bacterin, and 10 cows were injected with a placebo containing adjuvant only. Vaccine and placebo were administered at drying off, 30 d after drying off, and within 48 h after calving. Cows were challenged approximately 30 d after calving by intramammary infusion with a smooth heterologous strain of E. coli previously shown to cause mild clinical mastitis. Vaccination with the J5 bacterin reduced duration of IMI and local signs of clinical mastitis. Concentrations of BSA in milk 24 h after challenge were greater in control cows than in cows vaccinated with J5. The SCC at 7 d postchallenge were greater for cows vaccinated with the placebo than for cows vaccinated with J5. Bacterial counts were lower for cows vaccinated with the placebo than for cows vaccinated with J5 at 3, 6, and 9 h postchallenge. In contrast, cows vaccinated with J5 had lower bacterial counts at 2, 3, and 4 d postchallenge than did cows vaccinated with placebo. Systemic signs of clinical mastitis were relatively mild and similar between treatment groups. Rectal temperature, DMI, and milk production did not differ between control and cows vaccinated with J5 following challenge.