Purpose: This study investigated the association of serum lipid peroxidation (LPO) and glutathione peroxidase 4 (GPx4) with gestational diabetes mellitus (GDM) and metabolic abnormalities in Chinese pregnant women. Methods: The present case-control study was matched at a ratio of 1:1, and it recruited 132 pairs of participants at 24–28 gestational weeks. The serum LPO and GPx4 level were determined in each subject by enzyme-linked immunosorbent assay. The associations of LPO and GPx4 with metabolic parameters were analyzed. Thereafter, this study classified all subjects based on metabolic abnormality frequency (including body mass index, blood pressure, triglycerides, and fasting plasma glucose), and explored the association of the serum LPO and GPx4 levels in relation to metabolic abnormalities and clinical outcomes. Simultaneously, logistic regression analysis was used to estimate the odds radio (OR) and 95% confidence interval (CI) expressing the association between LPO/GPx4 and metabolic abnormalities. Results: Women with gestational diabetes mellitus (GDM) in second trimester displayed an increased LPO concentration, whereas the GPx4 concentration was decreased compared with normal subjects (174.58 \pm 22.01 ng/mL vs. 119.54 \pm 8.93 ng/mL, p 0.001 and 27.31 \pm 16.88 vs. 33.84 \pm 19.55 ng/mL, p 0.001, respectively). In addition, the GPx4 concentration was negatively associated with the plasma fasting 2 h plasma glucose level (2h-PG) and percentage glycated albumin (GA%) in the second trimester. Bivariate correlation analysis revealed that in GDM patients the serum GPx4 concentration displayed a significant linear correlation with glucose metabolism indexes, including fasting plasma glucose, glycated albumin, and 2h-PG (all p 0.05). By contrast, there was no relationship between the serum LPO concentration and glucose metabolism (p > 0.05) in GDM patients. Nevertheless, the LPO and GPx4 concentrations in the second trimester were not significantly related to the pregnancy/neonatal outcomes. Moreover, after the GDM subjects were grouped based on metabolic abnormality component, the metabolic abnormality risk was elevated with the increase in the LPO concentration (elevated diastolic blood pressure, OR = 1.04, p = 0.048; and high triglycerides, OR = 2.19, p 0.001), together with a greater incidence of multiple metabolic abnormalities. Additionally, the serum LPO concentration increased with the increased metabolic abnormality frequency (OR = 1.93, 95% CI: 1.62–2.29, p 0.001). Conclusions: In women with GDM, the serum GPx4 concentration was lower, which was strongly associated with second trimester glucose metabolism among the Chinese pregnant population. According to our findings, women with GDM had an increased LPO concentration, which was strongly associated with metabolic abnormalities among the pregnant women; this might be adopted as a predictor factor for metabolic abnormalities. The results of the present study suggest that a higher lipid oxidative stress and lower lipid antioxidant associated with an increased risk of GDM.