Abstract Objective Retinol-binding protein 4 (RBP4) is thought to be associated with insulin resistance in humans, while pregnancy is normally characterized by progressive insulin resistance. Gestational diabetes (GDM) occurs when pancreatic beta-cell function is unable to compensate for insulin resistance. This study aimed to determine whether or not serum RBP4 levels are elevated in pregnancy, and to explore the relationship between RBP4 levels and insulin resistance during pregnancy. Methods Serum RBP4 was measured at median gestational week 26 in 121 pregnant women, including 63 with GDM (GDM group) and 58 normal, glucose-tolerant pregnant women (P-NGT group), as well as 65 non-pregnant normal, glucose-tolerant women (NP-NGT group). Multiple stepwise regression analysis was used to explore the independent factors of RBP4. Results Serum RBP4 levels in the P-NGT and GDM groups were significantly higher than in the NP-NGT group (34.50 ± 9.80 mg/L and 41.64 ± 12.21 mg/L vs 30.64 ± 9.46 mg/L, respectively; P < 0.05) after adjusting for age, body mass index (BMI) and blood pressure. Furthermore, RBP4 levels were much higher in the GDM vs P-NGT group. Spearman's correlation analysis showed that serum RBP4 levels were positively correlated with triglycerides (TG), fasting plasma glucose, postprandial 2 h plasma glucose and HOMA-IR in pregnancy. Of these, TG and HOMA-IR ( r2 = 0.312) were independent factors of serum RBP4. Conclusion Serum RBP4 levels are significantly increased in pregnancy, independent of age and BMI, and are also considerably higher in pregnant women with GDM than in those with normal glucose tolerance. In addition, serum RBP4 levels appear to be a valuable marker of insulin resistance and dysfunctional lipid metabolism in pregnancy.