To assess the impact of maternal pre-pregnancy body mass index (BMI) on gestational diabetes mellitus (GDM) based on different pre-pregnancy hemoglobin levels. Methods: This retrospective cohort study included 1289 pregnant women between June 2020 and January 2022. Clinical data were collected by reviewing their medical and antepartum screening records between 24 and 28 gestational weeks, including pre-pregnancy BMI and pre-pregnancy hemoglobin (Hb) levels. The diagnosis of GDM mainly depended on oral glucose tolerance test (OGTT) during 24-28 weeks. Restricted cubic spline (RCS) was used to investigate the association between the pre-pregnancy Hb level and the risk of GDM. Univariate and multivariate logistic regression analyses were applied to evaluate the relative risk of GDM. Results: Of the 1289 included pregnant women, 187 (14.5%) women were diagnosed with GDM in this study. The pre-pregnancy Hb level was significantly associated with GDM risk, and the pre-pregnancy Hb level of 123 g/L was identified as the threshold to stratify and assess the association between the GDM risk and the pre-pregnancy BMI. For women with a pre-pregnancy Hb level ≥123 g/L, the pre-pregnancy BMI showed a significant association with GDM risk, and the estimated incidence rate of GDM was 7.7%, 14.8%, 36.3% and 44% for underweight, normal-weight, overweight and obese pregnant women, respectively. After adjusting for potential influencing factors of GDM, the respective relative risk was 1.0 (reference), 2.04 (95% CI 0.84, 4.99), 7. 06 (2.66, 18.61), and 10.77 (2.85, 40.63) (P for trend < 0.001). Conclusion:In pregnant women with a pre-pregnancy Hb level ≥123 g/L, pre-pregnancy BMI showed a more significant association with GDM risk as compared with those with a pre-pregnancy Hb level <123 g/L.