[Abstract] Objective: To investigate the effect of metformin on the content and composition of long-chain fatty acids (LCFA) in pregnant women with gestational diabetes mellitus (GDM) and their newborns. Methods: A total of 127 pregnant women who delivered in Changzhou Maternal and Child Health Hospital affiliated to Nanjing Medical University from August 2018 to August 2020 were selected, 32 pregnant women with normal glucose tolerance and 32 pregnant women with GDM who only needed diet control were selected as control group, and they were divided into N group and G1 group respectively. According to the principle of randomization and voluntariness, the pregnant women with GDM who needed drug treatment were randomly divided into metformin treatment group (G2 group, n = 33) and insulin treatment group (G3 group, n = 30). Blood glucose, blood lipids and insulin resistance index (HOMA-IR) were compared before delivery in each group. Body mass index (BMI) before pregnancy and delivery and pregnancy outcome were compared. The composition and content of long chain fatty acids in maternal blood and umbilical cord blood were analyzed. Results: The weight gain during pregnancy in G2 group was lower than that in N group, G1 group and G3 group, FBG in G2 and G3 group was higher than that in N group, 2HPG in G1, G2 and G3 group was higher than that in N group, HOMA-IR in G2 group was higher than that in N group, G1 group and G3 group (P < 0.05). There were no statistically significant differences in maternal blood TC, TG, FFA and neonatal weight among the four groups (P > 0.05). EA in G2 and G3 groups was higher than that in G1 group, while DPA in G2 group was higher than that in N group, G1 and G3 groups (P < 0.05). The contents of PA, OA, LA, ALA, EA, EPA, DPA, DPA6 and GLA in umbilical cord blood were lower than those in maternal blood (P < 0.05). The levels of PA, OA, EA, AA, EPA and DPA in maternal blood and umbilical cord blood were positively correlated, and the levels of PA, OA, LA, ALA, EA, AA, EPA, DHA and GLA in maternal blood were significantly negatively correlated with FBG (P < 0.05). Conclusion: Metformin and insulin have the same effect in the control of neonatal weight, blood glucose and blood lipid in the treatment of GDM, and metformin is better than insulin in the control of pregnancy weight. It has no significant effect on the contents of important fatty acids such as LA and ALA in maternal blood and umbilical cord blood, and has no significant effect on the contents of AA and DHA in umbilical cord blood. From the perspective of effect on essential fatty acids, the use of metformin in GDM is safe.