This experiment was conducted to study the effects of folic acid (FA) and betaine (BT) on lactation performance, milk fatty acid composition, nutrient digestion and rumen fermentation of dairy cows during perinatal period. Forty Holstein dairy cows during perinatal period with similar expected delivery date, parity, body weight and average milk yield during the previous lactation period were randomly divided into 4 groups with 10 cows in each group. Cows in the control group were fed a basal diet, and others in FA, BT and FA+ BT groups were fed basal diets supplemented with 135 mg/ d FA, 100 g/ d BT and 135 mg/ d FA+100 g/ d BT, respectively. The pre-experimental period lasted for 10 days, and the experimental period lasted for 42 days. The results showed as follows: 1) the actual milk, 4% fat-corrected milk, milk fat and milk protein yields, and milk fat rate, feed efficiency (FE) of FA and FA+BT groups were significantly higher than those of the control group (P<0.05). The 4% fat-corrected milk, milk fat and milk protein yields, and milk fat rate, FE of BT group were significantly higher than those of the control group (P<0.05). 2) The contents of C10∶0, C12∶0, C14∶0, C16∶1, C18∶3 and total 4 to 15 carbon fatty acid in milk fat of FA group were significantly higher than those of the control group (P<0.05), and the C18∶0 content was significantly lower than that of the control group (P<0.05). The contents of C10∶0, C12∶0, C14∶0, C16∶1, C18∶2, C18∶3 and total 4 to 15 carbon fatty acid in milk fat of BT and FA+BT groups were significantly higher than those of the control group (P<0.05), and the C18:0 and total >16 carbon fatty acid contents were significantly lower than those of the control group (P<0.05). 3) The apparent digestibilities of dry matter (DM), organic matter (OM), neutral detergent fiber (NDF) and acid detergent fiber (ADF) of FA, BT and FA+BT groups were significantly higher than those of the control group (P<0.05). 4) The rumen total volatile fatty acids (TVFA), acetate and butyrate contents and the ratio of acetate to propionate of FA, BT and FA+BT groups were significantly higher than those of the control group (P<0.05). In conclusion, the supplementation of FA and BT for dairy cows during perinatal period can improve the lactation performance and nutrient digestion, and the combined supplementation of FA and BT is more effective in improving lactation performance compared with than it addition alone.