Vitamin A and its derivatives contribute to many physiological processes, including vision, neural differentiation, and reproduction. Vitamin A deficiency causes early cessation of spermatogenesis, characterized by a marked depletion of germ cells. However, there has been no clear understanding about the role of chronic intake of vitamin A excess (VAE) in spermatogenesis. The objective of this study was to investigate whether chronic intake of VAE diet causes arrest of spermatogenesis. To examine the effects of VAE on spermatogenesis, we used ICR male mice fed with control (AIN-93G purified diet: 4 IU/g) diet or VAE (modified AIN-93G diet with VAE: 1,000 IU/g) diet for 7 weeks (from 3 to 10 weeks of age). At 10 weeks of age, the retinol concentration in the testes of VAE mice was significantly higher than that of control mice. Testicular cross sections from control mice contained a normal array of germ cells, while the seminiferous tubules from VAE mice exhibited varying degrees of testicular degeneration. Daily sperm production in VAE testes was dramatically decreased compared to that in control testes. Sperm viability, motility, and morphology were also impaired in VAE mice. Furthermore, we examined the effects of VAE on the expression of genes involved in retinoid signaling and spermatogenesis to determine the underlying molecular mechanisms. Therefore, we are the first to present results describing the long-term dietary intake of VAE impairs spermatogenesis using a mouse model.