To explore the correlation between the levels of serum 25-hydroxyvitamin D (25-OH-D), homocysteine (Hcy) and glycosylated hemoglobin (HbA1c) of pregnant women with gestational diabetes mellitus (GDM) during the first trimester of pregnancy and their insulin resistance. Methods: 134 pregnant women with GDM in hospital were included in study group from December 2019 to December 2022, and 76 normal pregnant women with the similar age and gestational weeks were selected in control group during the same period. The levels of blood lipids indicators, such as triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C), the levels of 25-OH-D, Hcy and HbA1c, and the values of insulin resistance indicators, such as fasting insulin (FINS), homeostasis model assessment of insulin resistance (HOMA-IR) and homeostasis model assessment of islet β cell function index (HOMA-β), of the women during 8-12 gestational weeks were compared between the two groups. Multivariate logistic regression was used to screen the risk factors of GDM occurrence. Pearson correlation analysis was adopted to analyze the correlation between the levels of 25-OH-D, Hcy and HbA1c of the pregnant women with GDM and their insulin resistance. Results: The levels of TG, TC and LDL-C of the women in the study group were significantly higher than those of the women in the control group, but the HDL-C level of the women in the study group was significantly lower. The 25 (OH) D level (25.89±2.67 nmmol/L) of the women in the study group was significantly lower than that (34.26±3.19 nmmol/L) of the women in the control group. The levels of Hcy (20.96±3.48μmol/L) and HbA1c (6.19%±0.31%) of the women in the study group were significantly higher than those (11.03±2.56μmol/L and 5.67%±0.97%) of the women in the control group, The values of FINS and HOMA-IR of the women in the study group were significantly higher than those of the women in the control group, but the HOMA-β value of the women in the study group was significantly lower. Multivariate analysis showed that the decrease of 25 (OH) D level, and the increases of Hcy, HbA1c and FINS level and HOMA-IR value of the women were the independent risk factors for their GDM occurrence. Pearson correlation analysis showed that the 25-OH-D level of the women with GDM was negatively correlated with their FINS level and HOMA-IR value, and was positively correlated with their HOMA-βvalue. The levels of Hcy and HbA1c of the women with GDM were positively correlated with their FINS level and HOMA-IR value, and were negatively correlated with their HOMA-β value (all P＜0.05). Conclusion: The levels of the serum 25-OH-D, Hcy and HbA1c of the pregnant women with GDM are closely related to their insulin resistance. Clinical monitoring the levels of 25-OH-D, Hcy and HbA1c and the insulin resistance situation of the pregnant women is beneficial to the prevention and treatment of their GDM during the first trimester of pregnancy.