Abstract: The aim of this study was to investigate the effects of different levels of supplementation with Hainanmycin on rumen fermentation parameters, methane production and microflora in vitro. The results showed that the addition of Hainanmycin could inhibit significantly the gas production (P<0.05) and the concentration of ammoniacal nitrogen (P<0.05), and pH enhanced linearly remarkably as the addition level of Hainanmycin increased (P<0.05). Compared to the control, the 10 mg·kg-1 Hainanmycin supplementation in the diet increased the pH by 4%. The content of propionate increased and the concentration of acetate, butyrate and A/P decreased significantly (P<0.05). The methane production was inhibited quadratically by the addition of Hainanmycin in the diet (P<0.05). The percentage of ruminococcu flavefaciens, fungi and protozoa accounting for the total bacterial 16S rDNA reduced significantly in experiment groups compared with the control (P<0.05), and methanogen and ruminococcus albus were not altered by the treatment. It was concluded that the supplementation of Hainanmycin in the diet could influence ruminal fermentation pattern, inhibit the methane production, and affect microflora significantly in vitro. And the methane production could reach the lowest level theoretically when the supplementation level of Hainanmycin was 7.2 mg·kg-1.