This study was carried out to examine the association between exposure to polycyclic aromatic hydrocarbon with traffic exhaust and biomarkers of lipid peroxidation and antioxidant levels among highway toll station workers. We conducted a cross-sectional study of 47 female highway toll station workers exposed to traffic exhausts and 27 female classroom trainees as a reference group. Exposure assessment was based on a biomarker of polycyclic aromatic hydrocarbon exposure, urinary 1-hydroxypyrene-glucuronide (1-OHPG). Urinary isoprostane was assayed as a biomarker of lipid peroxidation, and plasma antioxidative capacity of lipid-soluble substances (ACL) and water-soluble substances (ACW) was measured. The median concentration of urinary isoprostane was higher among the exposed non-smokers (4.63 ng/mL) compared with the reference non-smokers (3.52 ng/mL, difference: 0.91, 95% CI –0.15 to 1.98) (Wilcoxon rank-sum test: p = 0.04). The median concentration of ACW among non-smoking exposed subjects (37.9 μg/mL Trolox equivalent) was lower than that of the reference non-smokers (86.3 μg/mL). Adjusting for confounding effects by linear regression, a change in log(isoprostane) concentration was significantly related to a unit change in log(1-OHPG) (regression coefficient [ß], β = 0.14, 95% CI 0.07 to 0.21). Exposure to polycyclic aromatic hydrocarbon is associated with increased lipid peroxidation and reduced antioxidative capacity in toll station workers.