This study aimed to evaluate the effect of enzymatic hydrolysate of cottonseed protein (ECP) on the kinetic of gas production, rumen fermentation characteristics, and microbial diversity in continuous in vitro culture with a single factorial design of supplementation with various concentrations of ECP or yeast culture. Treatments were control (without supplementation, CON), supplementation with 10 g/kg Diamond-V XP yeast culture of substrate (XP), and supplementation with 6, 12 and 18 g/kg ECP of substrate (ECP1, ECP2, ECP3), each incubated with 30 mL of buffered incubation fluids and 200 mg of fermentation substrate in graduated glass syringes fitted with plungers for 48 h. Compared with the CON treatment, supplementation of XP yeast culture increased the cumulative gas production at 12 and 24 h, the concentration of ammonia nitrogen (NH3-N) concentration at 24 and 36 h, the concentration of microbial protein (MCP) concentration at 24 and 48 h, the molar butyrate proportion at 12, 24, and 48 h, the molar valerate proportion at 48 h, and the ratio of non-glucogenic to glucogenic acids (p < 0.05). Compared with the CON treatment, the concentration of MCP and the molar propionate proportion at 12 h were higher in the ECP1 treatment (p < 0.05); the cumulative gas production at 2, 4, and 12 h, the concentration of NH3-N at 36 h and the molar valerate proportion at 48 h were higher in the ECP2 treatment (p < 0.05); the cumulative gas production at 2, 12, and 48 h, the concentration of NH3-N at 12 and 36 h, the concentration of MCP at 12, 36, and 48 h, the molar butyrate proportion at 12 and 48 h, and the molar valerate proportion at 48 h were higher in the ECP3 treatment (p < 0.05). Compared with the CON treatment, supplementation with XP yeast culture significantly altered the relative abundance of the phyla Firmicutes, Kiritimatiellaeota, and Proteobacteria, while supplementation with ECP had minimal effect on bacterial diversity. The prediction of bacterial functions showed that the main gene functions of rumen bacteria are associated with carbohydrate metabolism, amino acid metabolism, and membrane transport. The findings of this study suggest that ECP can be used as a superior feed ingredient for ruminants, the suitable level of ECP was 18 g/kg in vitro experiment.