Although exposure to environmental toxicants, including endocrine-disrupting chemicals, is thought to be a possible cause of male infertility, the pathogenesis of male reproductive disorders remains unclear. In the present study, we used Gulo-/- mutant mice, which are unable to synthesize ascorbic acid, to study the importance of dietary vitamin C (VC) on spermatogenesis. Regular chow containing approximately 110 mg/kg VC is unable to support the growth of these mutant mice, but a VC supplement in their drinking water (330 mg/L) is able to ameliorate the VC deficiency. Testes of Gulo-/- mutants born from heterozygous mothers without VC supplement (VC-deficient mice) and those born from mothers given a VC supplement (VC-sufficient mice) were examined by morphological and biochemical analyses. Morphological analysis revealed that apoptosis of spermatocytes occurred frequently in VC-deficient mice at 20 days of age. Two-dimensional electrophoresis analysis revealed the specific disappearance of heat-shock protein (Hsp) 70 in the testes of 20-day-old VC-deficient mice. In the present study, the relationship between the apoptosis of spermatocytes and Hsp70 in VC-deficient mice is discussed.