An in vitro fermentation was conducted to determine the effects of hainanmycin on protein degradation and populations of ammonia-producing bacteria. The substrates (DM basis) for in vitro fermentation consisted of alfalfa hay (31.7%), Chinese wild rye grass hay (28.3%), ground corn grain (24.5%), soybean meal (15.5%) with a forage: concentrate of 60:40. Treatments were the control (no additive) and hainanmycin supplemented at 0.1 (H0.1), 1 (H1), 10 (H10), and 100 mg/kg (H100) of the substrates. After 24 h of fermentation, the highest addition level of hainanmycin decreased total VFA concentration and increased the final pH. The high addition level of hainanmycin (H1, H10, and H100) reduced (p<0.05) branched-chain VFA concentration, the molar proportion of acetate and butyrate, and ratio of acetate to propionate; and increased the molar proportion of propionate, except that for H1 the in molar proportion of acetate and isobutyrate was not changed (p>0.05). After 24 h of fermentation, H10 and H100 increased (p<0.05) concentrations of peptide nitrogen and AA nitrogen and proteinase activity, and decreased (p<0.05) $NH_3$-N concentration and deaminase activity compared with control. Peptidase activitives were not affected by hainanmycin. Hainanmycin supplementation only inhibited the growth of Butyrivibrio fibrisolvens, which is one of the species of low deaminative activity. Hainanmycin supplementation also decreased (p<0.05) relative population sizes of hyper-ammonia-producing species, except for H0.1 on Clostridium aminophilum. It was concluded that dietary supplementation with hainanmycin could improve ruminal fermentation and modify protein degradation by changing population size of ammonia-producing bacteria in vitro; and the addition level of 10 mg/kg appeared to achieve the best results.