Although varicocele is a relatively common entity encountered in the evaluation of infertile men, the exact pathophysiology still remains unclear. Recently, as previously widely investigated in various parts of human circulatory system, nitric oxide synthase (NOS) and its product, nitric oxide (NO) have been thought to play a role in the development of varicocele and thus male infertility. In this study, we determined the concentration of NO metabolite and the expression of NOS isoforms in the internal spermatic (ISV) and superficial branch of inferior epigastric veins of infertile men with varicocele. The study included 60 infertile men with clinically unilateral or bilateral varicocele. Expression of inducible and endothelial NOS (iNOS and eNOS) isoforms were investigated in tissue arrays of internal spermatic and superficial branch of inferior epigastric veins with immunohistochemistry. NO metabolite (nitrite) levels were measured using the calorimetric method. A significantly higher expression of eNOS was observed in the varicose veins (mean score: 2.25 and 1.55, respectively; p = 0.0001). However, statistically, there was no significant difference for expression of iNOS between varicose and control veins (p = 0.094). The nitrite concentration and NOS expression were not found to be correlated with clinical variables (varicocele grade, maximum varicose vein diameter, and sperm concentration, motility, and morphology) (p > 0.05). As a result, the significantly higher expression of eNOS in ISV may be responsible for the development of varicocele, although this finding is not accompanied by an increase in NO concentration. Still, the pattern of the relationship between varicocele and increased eNOS expression warrants further investigation.