The purpose of this research was to investigate the effects of replacing soybean meal (SBM) with a fermented corn gluten-wheat bran mixture (FCWM) on nutrient digestibility, lactation performance, plasma metabolites, ruminal fermentation, and bacterial communities in Holstein cows. Nine healthy multiparous (parity = 3) Holstein cows with similar body weights (624 ± 14.4 kg), days in milk (112 ± 4.2), and milk yields (31.8 ± 1.73 kg; all mean ± standard deviation) were used in a replicated 3 × 3 Latin square design with 3 periods of 28 d. Cows were fed 1 of 3 dietary treatments in which FCWM replaced SBM as follows: basal diet with no replacement (0FCWM); 50% replacement of SBM with FCWM (50%FCWM); and 100% replacement of SBM with FCWM (100%FCWM). The diets were formulated to be isocaloric and isonitrogenous. The results showed that the total-tract digestibility of dry matter and crude protein increased linearly with increased dietary FCWM, and we found a trend for increased total-tract neutral detergent fiber and potentially digestible NDF digestibility. Milk yield tended to increase in a linear manner as more FCWM was consumed, and energycorrected milk production was significantly increased with FCWM supplementation as a result of increased milk protein and lactose yields. Plasma glucose and IgG concentrations increased linearly with increasing FCWM supplementation, but plasma malondialdehyde concentration decreased linearly. Concentrations of total volatile fatty acids and propionate showed a linear increase with increasing FCWM supplementation, leading to a linear decrease in pH. The relative abundance of ruminal Prevotellaceae, Veillonellaceae, and Prevotella 1 increased linearly with increasing FCWM supplementation, and the relative abundance of ruminal Succinivibrionaceae and Muribaculaceae decreased linearly.The relative abundance of fecal Ruminococcaceae, Prevotellaceae, and Ruminococcaceae UCG-005 increased linearly with increasing FCWM supplementation, but the relative abundance of fecal Peptostreptococcaceae decreased linearly. Overall, the replacement of SBM with FCWM altered the composition of the ruminal bacterial community and improved nutrient digestibility, lactation performance, and ruminal fermentation in cows, providing a data reference for the use of FCWM in dairy production.