Previous studies have uncovered a progestin-only contraceptive association with an increased risk of diabetes, but limited study has explored the relationship of endogenous progesterone and pregnenolone level with diabetes status. A case-control study was conducted in Henan Rural Cohort (Register Number: ChiCTR-OOC-15006699), to evaluate the dose-response independent and interactive relationship of progesterone and pregnenolone levels with prediabetes and type 2 diabetes mellitus (T2DM) in Chinese rural population.A case-control study.A total of 798 T2DM patients, 779 prediabetes patients, and 782 individuals with normal fasting plasma glucose were included in this study. Serum progesterone and pregnenolone were detected by liquid chromatography-tandem mass spectrometry. Logistic regression and restricted cubic splines were used to assess the independent effects of progesterone and pregnenolone on prediabetes and T2DM. Interactive plots were employed to examine the interaction effects of progesterone and pregnenolone.Progesterone in the fourth vs first quartile was positively associated with prediabetes (odds ratio (OR) [95% confidence interval (CI)]: 2.66 [1.99-3.55]) and T2DM (OR [95%CI]: 6.41 [4.57-8.98]), whereas, pregnenolone in the fourth vs first quartile was inversely related to prediabetes (OR [95%CI]: 0.23 [0.16-0.33]) and T2DM (OR [95%CI]: 0.44 [0.31-0.62]). Additionally, the nonlinear dose-response associations between progesterone and pregnenolone with prediabetes and T2DM were found. Interactive effects of progesterone and pregnenolone on prediabetes and T2DM were observed, and these significant associations remained in gender-stratified analysis.Prediabetes and T2DM were positively linked to serum concentration of progesterone, and negatively related to pregnenolone in dose-response manner in Chinese rural population.