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Objective Metallothionein (MT), a metal-binding protein, protects cells against apoptosis under hypoxia. Hypoxic stress causes pathophysiologic changes in the internal spermatic vein (ISV) of patients with varicocele. The use of confocal microscopy and fluorescent compounds permits visualization of the vascular wall organization and molecular changes that may occur. We studied MT expression and distribution in the ISV using confocal laser scanning microscopy. Materials and Methods The study group consisted of 20 patients with grade 3 left varicocele. The control group consisted of 10 volunteers with left indirect inguinal hernia. Through a left inguinal incision, a 1-cm section of the ISV was resected from each patient to detect MT expression by immunohistochemical stain and confocal microscopy. Results Immunohistochemical analysis of MT in the ISV sections showed stronger staining in the varicocele group than in the control group. The use of confocal microscopy provided a detailed vascular wall structure, and also revealed that the MT protein was distributed significantly in the endothelium. Conclusion The hypoxic conditions induced the overexpression of MT, which is deposited in the endothelial layer of varicocele veins, as determined by confocal microscopy. MT has the function to protect vascular cells from apoptosis, and this may be a mechanism for the thickened wall of the ISV that is present in varicocele patients.