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The purpose of the paper was to study the effects of cellulolytic bacteria (CB) mixed with nitrogen-fixing bacteria (NFB) on fermentation in vitro. Nine CB strains and seven NFB strains were isolated from rumen fluid of three Holstein cows. Based on higher activity of cellulose or nitrogenase, three CB types [CB-2(KT725624), CB-5(KT725623) and CB-6(KT725622)] and one NFB type [NFB-3(KT735054)] were screened out, respectively. Two parts were included in this study. The first part explored the optimal mixed ratio of CB to NFB through inoculating filter paper medium with the bacteria of 2×105 cfu ? mL-1. According to CMCase and FPase activities in the medium, the ratio of 4 to 1 was proven to be more effective. In the second part, rumen fermentation in vitro was conducted at 4 : 1 of CB to NFB, aiming at studying the effects of mixed bacteria. Six groups were classified, namely, control group (no bacteria), Group 1 (CB-2+NFB-3), Group 2 (CB-5+NFB-3), Group 3 (CB-6+NFB-3), Group 4 (NFB-3) and Group 5 (CB-6). All the experimental groups had the same amount of bacteria (4×106 cfu ? mL-1) in the fermentation liquid. Samples were collected at 2, 4, 8, 12 and 24 h of incubation. Compared with the groups with CB or NFB alone, gas production, dry matter degradability and bacterial protein expressions in the mixed groups increased. However, NH3-N concentration decreased and pH was stable. Meanwhile, related values among three mixed groups were significantly different; values in Group 2 were the best.