In this study, we evaluate the effects of chitosan level for cows in late lactation on ruminal fermentation and milk yield and composition. Eight Holstein cows cannulated in the rumen (215.4 ± 60.9 DIM, 22.07 ± 5.32 kg of milk yield and 641.6 ± 41.06 kg of BW) in replicated 4 × 4 Latin squares, were fed the following diets: (CTR) control diet without addition of chitosan, CHI75 mg/kg, CHI150 mg/kg and 225 mg/ kg of chitosan addition per kg BW. Each period had 14 d adaptation and 7 for collection data. Ruminal fluid was collected on 20th day of each period at 7 times to evaluate the effect of the diets on ruminal fermentation. Sampling of milk was done on 16 to 18th day of each period to evaluate the composition. The results of milk composition was subjected to ANOVA, while ruminal fermentation data were analyzed as repeated measures by PROC MIXED of SAS. Chitosan changed ruminal fermentation profile, increasing rumen propionate (P < 0.05) without shift acetate and total VFA concentrations (P > 0.05). However, chitosan decreased linearly acetate:propionate ratio (P < 0.05) similarly to butyrate and AGCR concentrations (P < 0.05). Chitosan increased milk yield (P < 0.05). Furthermore, dietary chitosan linear increased protein and lactose milk production (P < 0.05). Chitosan as feed additive improve animal performance and ruminal fermentation with increased ruminal propionate concentration.