The objective of this study was to evaluate the influence of supplemental flavomycin on cattle growth performance, carcass characteristics, diet digestibility, and ruminal fermentation characteristics of calf-fed Holstein steers. One hundred Holstein steers (123 ± 7 kg) were balanced by weight and assigned to 20 pens. Dietary treatments consisted of a steam flaked corn-based diet supplemented with (dry matter basis): 1) control, no feed additive; 2) 6.6 mg/kg flavomycin; 3) 13.2 mg/kg flavomycin, and 4) 30 mg/kg monensin (MON). There were no treatment effects (P ≥ 0.17) on live weight, average daily gain (ADG), and gain efficiency. Flavomycin did not affect dry matter intake (DMI; P ≥ 0.24). Flavomycin supplementation did not affect (P ≥ 0.37) the ratio of observed vs. expected DMI. However, MON decreased (P = 0.02) observed vs. expected DMI by 3.7%. There were no treatment effects (P ≥ 0.44) on ruminal pH or temperature. Flavomycin did not affect (P ≥ 0.13) carcass characteristics and liver abscess among steers. Four Holstein steers (463 ± 20 kg) with ruminal cannulas were used in 4 × 4 Latin square experiment to study treatment effects on site and extent of digestion, ruminal pH, and volatile fatty acid (VFA) molar proportions. Dietary treatments were the same as experiment 1. Flavomycin tended to increase (linear effect, P = 0.07) ruminal organic matter (OM) digestion, associated with increased (linear effect, P < 0.01) ruminal starch digestion. Supplementing flavomycin at 13.2 mg/kg decreased net microbial N synthesis (quadratic effect, P = 0.03). Compared with control, MON tended to increase (P = 0.10) ruminal neutral detergent fiber (NDF) digestion and increased (P < 0.01) ruminal starch digestion. Monensin did not affect (P = 0.39) net microbial N synthesis, but decreased (P = 0.01) ruminal degradation of feed nitrogen (N). There were no treatment effects (P > 0.10) on total tract apparent digestion of DM, OM, NDF, and starch. Flavomycin decreased ruminal pH (quadratic effect, P < 0.01) measured 4 h postprandial. Compared with control, MON increased ruminal pH (P = 0.03). Flavomycin increased (linear effect, P = 0.03) ruminal propionate molar proportion and decreased (linear effect, P ≤ 0.04) ruminal molar proportions of acetate and butyrate, and decreased (linear effect, P = 0.02) acetate:propionate molar ratio and estimated methane production. We conclude that supplementing flavomycin at 6.6 or 13.2 mg/kg had no major effects on cattle growth performance, carcass characteristics, diet digestibility, and ruminal fermentation characteristics.