Bisphenol A (BPA) is a pervasive environmental toxicant with known reproductive effects on sperm parameters and hormone levels. Several observational studies have investigated the associations between BPA exposure and male reproductive function, but findings are inconsistent. The objective of this study was to assess the associations between urinary BPA concentrations and semen quality and reproductive hormone levels in a cross-sectional study with 215 healthy young university students (18–23 years old), investigated between 2010 and 2011 in Southern Spain (Murcia Region). All subjects provided urine, blood serum and semen samples on a single day. Urinary BPA concentrations were measured by dispersive liquid–liquid microextraction and ultra-high performance liquid chromatography with tandem mass spectrometry detection. Semen quality was evaluated by measuring volume, concentration, motility, morphology and total sperm count (TSC). Serum samples were analyzed for reproductive hormones, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, inhibin B and estradiol. Relationships between urinary BPA concentrations and semen quality parameters and reproductive hormone levels were examined using linear regression, adjusting for potential confounders and covariates. Ninety-five percent of the men had detectable urinary BPA concentrations with unadjusted median (5th–95th) of 2.8 (0.16–11.5) ng/mL. After adjustment for important covariates, there was a significant positive association between urinary BPA concentrations and serum LH levels (β = 0.07, 95%CI: 0.02;0.12, p-value < 0.01). Urinary BPA concentration was also significantly and inversely associated with sperm concentration (β = − 0.04, 95%CI: − 0.07;− 0.02, p-value < 0.01) and TSC (β = − 0.05, 95%CI: − 0.08;− 0.02, p-value < 0.01). No significant associations were found between BPA and other semen parameters or reproductive hormone levels. Our results support the hypothesis that BPA exposure may be associated with a reduction in Leydig cell capacity (increased LH levels) and decreased sperm counts in young men.