[Objectives] The research on mammalian sexual reproduction is beneficial to the exploration of human reproduction mechanism, which focuses on reproductive system, germ cells, fertilization cells, external environmental factors, etc. There is little research on mammalian fertility after the elimination of metabolic enzymes from the body. It is known that aldehyde dehydrogenase 2 (ALDH2) is the most important enzyme for aldehyde oxidation, which can metabolize a large number of harmful aldehydes produced in the body. Studies have shown that a large number of reactive oxygen species produced by male sperm during exercise can increase the aldehyde content in sperm. Therefore, we studied the fertility of ALDH2 gene knockout male mice. [Methods] In this study, we analyze the testicular organ coefficient, testicular tissue cell morphology, sperm motility parameters, the number of offspring born after mating, and the ratio of male to female offspring of 52 male C57BL/6 mice of different weeks of age with ALDH2 gene knockout. Mean value comparison, one-way ANOVA, and t-test implemented in SPSS were used to explore the effect of ALDH2 gene knockout on fertility of male mice. [Results] We found that compared with the wild type (WT) mice, the weight and testicular organ coefficient of knockout (KO) mice at 5 and 10 weeks of age and mice at 3 and 10 weeks of age had extremely significant differences (n = 6, P < 0.01) (Table 1), the weight of mice at 3 and 7 weeks of age and testicular organ coefficient of mice at 5 and 7 weeks of age had significant differences (n = 6, P < 0.05) (Table 1). Among them, the cytoplasm of testicular tissue of 10 week old ALDH2 gene knockout male mice became larger (Fig. 2b); the sperm activity rate of knockout mice was significantly reduced (n = 6, P < 0.05) (Table 2); the litter size of ALDH2 transgenic mice was significantly reduced (6.7 ± 0.5) compared with wild type mice (11.0 ± 2.0, n = 3, P < 0.05), and the female to male ratio of ALDH2 gene knockout mice was also significantly reduced (0.8 ± 0.1 vs. 1.8 ± 0.5, n = 3, P < 0.05). [Conclusion] This study provides a basis for revealing the role of ALDH2 gene in male mouse reproduction and provides valuable information for the potential diagnosis and in vitro application of assisted reproductive technology.