Background: Experimental evidence suggested that per- and polyfluoroalkyl substances (PFAS) may affect the production of reproductive and sex steroid hormones. However, epidemiologic evidence regarding the effects of in utero exposure to PFAS, particularly to short-chained PFAS, on sex hormones in fetus are scarce and inconsistent. Objective: This study aimed to assess the relationship between maternal PFAS exposure and reproductive and sex steroid hormones in the fetal umbilical cord blood. Methods: A total of 752 pregnant women who were recruited in the Shanghai Birth Cohort Study between 2013 and 2016 were selected. We quantified 9 PFAS in early pregnancy (median gestational age at 12 weeks) in maternal blood using HPLC-MS/MS. Dehydroepiandrosterone sulfate (DHEA-S), sex hormone-binding globulin (SHBG), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone (T) were measured in the cord blood using chemiluminescence kits. Both PFAS concentrations and hormones were ln-transformed due to their right skewed distribution. Multiple linear regression was used to evaluate the associations between PFAS and hormones indicators. Results: One ln- unit increase in maternal perfluorobutane sulfonate (PFBS) was related to decreased FSH [-0.093 (95% CI: -0.170, -0.016)] and LH [-0.099 (95% CI: -0.199, -0.001)], and increased SHBG [0.040 (95% CI: 0.007, 0.074)] in cord blood. These associations were larger in girls but non-significant in boys. In girls, FSH (-0.126, 95% CI: -0.244, -0.009), LH (-0.147, 95% CI: -0.301, 0.008) and SHBG (0.062, 95% CI: 0.003, 0.122). No association was found for other 8 PFAS. Conclusions: Prenatal exposure to PFBS was associated with the interruption of fetal reproductive and sex hormone homeostasis in this prospective cohort. These significant associations were limited to girls. Our findings suggested that reproductive toxicity of short-chain PFAS may not be neglected.