Most of the general populations are exposed to polycyclic aromatic hydrocarbons (PAHs) at different levels. A limited number of studies have suggested that PAHs exposure may be associated with semen quality. To examine the association of four PAH metabolites, 1-hydroxynapthalene (1-N), 2-hydroxynapthalene (2-N), 1-hydroxypyrene (1-OHP) and 2-hydroxyfluorene (2-OHF) with altered semen quality, 542 subjects were recruited through the clinic following strict eligibility screening. Using LC-MS/MS, individual exposures were measured as spot urinary concentrations of PAH metabolites adjusted by creatinine (CR). Semen quality was assessed by semen volume, sperm concentration, sperm number per ejaculum, and sperm motility. First, we confirmed the variability of PAH metabolites in human urine. Our results showed that the median CR-adjusted concentrations of 1-N, 2-N, 1-OHP, 2-OHF were 2.35, 4.05, 1.14, and 2.89 microg/g of CR, respectively. Significant P-values for trend were found that men with higher 1-OHP (assessed as quintiles) were more likely to have below-reference sperm concentration and sperm number per ejaculum. These results indicate that PAHs exposure might be related to altered human semen quality.