Colicin E1 (ColE1) is a bacteriocin produced by and effective against Escherichia coli and related species. The current study examined ColE1 as a potential intervention strategy for controlling E. coli O157:H7 contamination on beef carcasses. Untrimmed beef round roasts were cut into sample sizes of 5.08 by 2.52 by 5.08 cm, with an adipose layer covering an entire surface of lean beef. Samples were placed on sterile metal hooks and inoculated with E. coli O157:H7 at a level of 5 log CFU/ml in sterile tryptic soy broth. After inoculum attachment, ColE1 in doses of 0, 100 μg, 500 μg, and 1 mg/ml of 10mM Tris, pH 7.6, was sprayed on the samples for a period of 10 min. Samples were evaluated at 0 and 30 min, 1, 2, 3, 4, and 5 days post-spraying at 10°C for E. coli O157:H7 inhibition. Treating samples with 500 μg and 1 mg of ColE1 effectively inhibited E. coli O157:H7 growth. When these doses were applied to samples inoculated with E. coli WS 3331, E. coli contamination was reduced by 4 and 7 log CFU/cm2, respectively, compared with the untreated control samples. In strain WS 3331, treatment with 1 mg ColE1 significantly inhibited growth of E. coli O157:H7 compared with the untreated control during the entire study. ColE1 provided powerful reduction of E. coli O157:H7 as a beef carcass spray intervention.