Interest in the high folate status of pregnant women has increased due to its role in the prevention of neural tube defects (NTDs). The effect of increased red blood cell (RBC) folate status during the second trimester of pregnancy on gestational diabetes mellitus (GDM) remains unclear. We measured RBC folate concentrations by competitive protein‐binding assay and obtained clinical information from electronic medical records. Logistic regression analysis was used to explore the associations of RBC folate concentrations with risks of gestational diabetes mellitus (GDM). We further assessed the potential nonlinear relations between continuous log‐transformed RBC folate concentrations and GDM risk by using the restricted cubic splines. We observed high RBC folate concentrations in GDM patients compared to control group [median (interquartile range, IQR), GDM vs. controls: 1,554.03 (1,240.54–1,949.99) vs. 1,478.83 (1,124.60–1,865.71) nmol/L, p = .001]. Notably, high folate concentrations were significantly associated with an increased risk of GDM [RR per 1‐SD increase: 1.16 (1.03, 1.30), p = .012] after adjustment for maternal age, parity, and body mass index (BMI) at enrollment. In the restricted cubic spline model, a test of the null hypothesis of the linear relationship was rejected (p = .001). Our study firstly showed that maternal RBC folate concentrations during the second trimester of pregnancy increase the risk of GDM in a Chinese population. Further randomized clinical trials (RCTs) are warranted to confirm the adverse effect.