The objective of the current experiment was to elucidate the effect of peptides enzymatic hydrolysis from cottonseed protein on rumen microbial population, and hence on milking performance of lactating dairy cows. A total of 50 Holstein cows were assigned into two groups, fed diets with or without enzyme hydrolyzed cottonseed protein (ECP) supplementation. The supplemented group received 0.4% ECP in the diet. Results of rumen microbial population showed that ECP supplementation had no effect on the overall microbe diversity but an increasing trend in the population of Succiniclasticum spp. was observed with ECP supplementation (P>0.05). The results of milk production and composition showed higher lactose and milk yields in ECP supplemented diet as compared to the control group (P<0.05). However, no significant difference was observed in milk protein, milk fat, and milk urea nitrogen between the two groups (P>0.05). Dry matter, neutral detergent fiber and acid detergent fiber digestibility was also observed to be higher in the ECP group (P<0.05), while CP digestibility was not affected by ECP supplementation (P>0.05). Furthermore, an increased blood total protein and glucose and a decreased blood urea nitrogen were observed in the ECP group (P<0.05). In conclusion, it is suggested that the ECP is a rich source of peptides that can be used as a feed supplement for rumen microbial proliferation, improvement of rumen fermentation, and increase in milk yield.