Chitosan, a non-toxic and biodegradable carbohydrate polymer, had diverse potential applications in the food processing, pharmaceutical, cosmetics, veterinary, environmental protection, agricultural industries and medicine. However, there is limited knowledge with regard to the different dose of chitosan in ruminant nutrition and whether chitosan is related to the altered bacterial community is still unclear. A completely randomized design was applied in vitro incubation. A basal diet with no chitosan was the control, and the treatment diets were supplemented with chitosan at the dose of 0.4, 0.8, 1.2, 1.6, and 2.0% DM. Six fermentation units were used per treatments. The concentrations of methane and volatile fatty acids were determined using a gas chromatograph. To explore methane inhibition following fermentation and the response of ruminal microbiota to different dose of chitosan, the bacterial 16S rRNA genes were sequenced by Miseq high-throughput sequencing and were confirmed by realtime-PCR. The results revealed that the dose of 2.0% chitosan had greatest reduction in methane emission and shifted the fermentation pattern toward propionate production. Real-time PCR analysis showed that chitosan increased the population of Fibrobacter succinogenes and Megasphaera spp. relative to total bacterial 16S rDNA, while the dose of 2.0% CHI significantly increased the population of Ruminococcus albus compared with control (P < 0.01). Furthermore, 0.4% CHI could significantly increase the population of Succinivibro spp. (P < 0.01), but 2.0% CHI had no effect. ANOSIM showed no significant differences in bacterial community composition among all the treatments (R = −0.03, P = 0.71). Chitosan addition had a tendency of increase the feed digestibility and significantly increased the VFA production (P < 0.10). In particular, correlation analyses between relative abundances of bacteria genera showed that the propionate concentration was positively related with Prevotellaceae and Bacteroidales (P < 0.05), but negatively correlated with Ruminococcacease (P < 0.05). Therefore, the effects of chitosan on microbial populations and bacterial communities, particularly on propionate production, are probably responsible for the discrepancy in its effects on rumen fermentation and methane emission, and thus it may be used as a potential alternative to fed to ruminants.