The study evaluated the effects of folic acid （FA） on rumen fermentation parameters， antioxidant indicators and microbial population in dairy cows in vitro. Four early lactation Holstein dairy cows were used as the donor of ruminal fluid， and the total mixed ratio of dairy cows was used as the fermentation substrate. The supplementation level of FA was 0， 1.5， 3.0， 4.5， 6.0， 7.5 and 9.0 mg·kg^-1 dry matter （DM）， respectively. After incubation at 39℃ for 24 h， ruminal DM degradability （DMD）， gas production （GP）， FA content， antioxidant indicators， fermentation parameters， microbial enzyme activity and population were determined. The results were showed as follows： 1） The DMD was greater （P<0.05） for the supplementation of FA at 1.5， 3.0 and 4.5 mg·kg^-1 DM， and the 24 h GP and theoretical maximum GP were greater （P<0.05） for the supplementation of FA at 1.5， 3.0， 4.5 and 6.0 mg·kg^-1 DM compared with other groups. 2） Ruminal fluid FA content was greater （P<0.05） and malondialdehyde content was lower （P<0.05） for the groups with FA supplementation compared with the control. The activity of superoxide dismutase was greater （P<0.05） in the groups with 1.5， 3.0， 4.5 and 6.0 mg·kg^-1 DM FA supplementation compared with other groups. The activity of glutathione peroxidase was greater （P<0.05） in the groups with supplementation of FA at 3.0， 4.5， 6.0 and 7.5 mg·kg^-1 DM than in the control and 1.5 mg·kg^-1 DM FA supplementation groups. The total antioxidant capacity activity was greater （P<0.05） for the supplementation of FA at 1.5， 3.0， 4.5， 6.0 and 7.5 mg·kg^-1 DM than for the control. 3） The groups with FA addition had greater （P<0.05） total volatile fatty acids （TVFA） and acetate production compared with the control， and the production of TVFA and acetate were greater （P<0.05） for the 3.0 and 4.5 mg·kg^-1 DM FA supplementation than for 7.5 and 9.0 mg·kg^-1 DM FA supplementation. 4） The groups with FA addition had greater （P<0.05） activities of cellobiase and xylanase compared with the control， and the activity of cellobiase was greater （P<0.05） for the supplementation of FA at 3.0， 4.5 and 6.0 mg·kg^-1 DM than for the supplementation of FA 1.5 and 7.5 mg·kg^-1 DM， and the activity of xylanase was greater （P<0.05） for the 1.5， 3.0 and 4.5 mg·kg^-1 DM FA supplementation than for the 7.5 and 9.0 mg·kg^-1 DM FA supplementation. 5） The groups with FA addition had greater （P<0.05） populations of total bacteria， protozoa and R. flavefaciens compared with the control. The total bacteria population was greater （P<0.05） in the 4.5， 6.0 and 7.5 mg·kg^-1 DM FA supplementation groups than in the 1.5 and 9.0 mg·kg^-1 DM FA supplementation groups. The R. flavefaciens population was greater （P<0.05） for the 1.5， 3.0 and 4.5 mg·kg^-1 DM FA supplementation than for the 7.5 and 9.0 mg·kg^-1 DM FA supplementation. The fungi population was greater （P<0.05） in the 3.0， 4.5， 6.0 and 7.5 mg·kg^-1 DM FA groups than in the control， 1.5 and 9.0 mg·kg^-1 DM FA groups. The R. albus population was greater （P<0.05） for the groups with 1.5， 3.0， 4.5 and 6.0 mg·kg^-1 DM FA supplementation than for the control， 7.5 and 9.0 mg·kg^-1 DM FA groups. The F. succinogenes population was greater （P<0.05） in the 4.5， 6.0， 7.5 and 9.0 mg·kg^-1 DM FA groups than in the control and 1.5 mg·kg^-1 DM FA group. In conclusion， the supplementation of FA improves rumen antioxidant status， stimulate cellulolytic and amylolytic bacteria growth， and resulting in an increase in DMD and TVFA production. The proper level of ruminal FA supply is 1.5-3.0 mg·kg^-1 DM in the condition of the current study.