BackgroundCytochrome P450 2E1 (CYP2E1) plays a key role in the metabolism of xenobiotic and endogenous low-molecular-weight compounds. This study aimed to determine if the genetic variations of 96-bp insertion/deletion (I/D) and C-1054T (rs2031920) inCYP2E1were associated with the risk of gestational diabetes mellitus (GDM).MethodsCYP2E1polymorphisms were genotyped in a case-control study of 1,134 women with uncomplicated pregnancies and 723 women with GDM. The effects of genotype on the clinical, metabolic, and oxidative stress indices were assessed.ResultsTheCYP2E1C-1054T variant was associated with an increased risk of GDM based on the genotype, recessive, dominant, and allele genetic models (P< 0.05). The TT + CT genotype remained a significant predictive factor for GDM risk after correcting for maternal age and pre-pregnancy body mass index (OR = 1.277, 95% CI: 1.042–1.563,P= 0.018). Moreover, fasting insulin concentrations and homeostatic model assessment of insulin resistance were significantly higher in GDM patients carrying the T allele than in those with the CC genotype (P< 0.05). Furthermore, the combined genotype II + ID/TT + CT of the 96-bp I/D and C-1054T polymorphisms further increased the risk of GDM when the combined genotype DD/CC was set as the reference category (OR = 1.676, 95% CI: 1.182–2.376,P= 0.004).ConclusionsThe T allele of the C-1054T polymorphism and its combination with the I allele of the 96-bp I/D variation inCYP2E1are associated with an increased risk of GDM in the Chinese population. The − 1054T allele may be associated with more serious insulin resistance in patients.