Objectives The effects of adding rumen-protected folic acid to dietary on the pregnancy rates, levels of reproductive hormones including estrogen and progesterone in plasma, and indexes of folic acid metabolism in Holstein dairy cows were studied to provide the scientific basis for efficiently utilizing folic acid in Holstein dairy cows. Methods 160 multiparous Holstein dairy cows with similar physical condition, parity, and lactation days were randomly divided into 4 groups, with 40 dairy cows in each group. The dairy cows were fed with a total mixed diet supplemented with 0 g, 3 g, 6 g, and 9 g rumen-protected folic acid during the period from 17 days before artificial insemination to 35 days after insemination. The ultrasound examination was conducted on the experimental dairy cows on the 35th day of artificial insemination to count the conception rate of the first and second mating. The blood was collected to measure the content of estrogen (E₂), progesterone (P₄), folic acid, and vitamin B₁₂ in plasma. Results The conception rate of the first mating, the content of E₂ and folic acid in plasma in the group fed with 3 g rumen-protected folic acid was significantly higher than that in the control group fed with 0 g rumen-protected folic acid(P<0.05). There was no significant difference in the conception rate of the second mating, and the content of P₄ and vitamin B₁₂ in plasma among the groups(P>0.05). Conclusions It is indicated that adding 3 g rumen-protected folic acid to dietary can increase the conception rate of the first mating and the content of E₂ and folic acid in plasma in Holstein dairy cows under the conditions of this experiment.