Sleep deprivation (SD), a prevalent modern lifestyle issue, has been associated with impaired male reproductive function, yet the underlying molecular mechanisms remain poorly understood. In a mouse model, sustained SD triggered germ cell apoptosis, reduced sperm motility, and increased morphological abnormalities. Furthermore, SD compromised sperm fertilization capacity and impaired preimplantation embryo development. Proteomic analysis identified SLC46A1, a proton-coupled folate transporter, as the only consistently downregulated protein in both testicular tissue and sperm. Furthermore, the expression of methylenetetrahydrofolate reductase (MTHFR), a key enzyme in folate metabolism, was also markedly decreased, accompanied by reduced levels of core DNA repair proteins (RAD51 and TOPBP1). Notably, folate supplementation prior to and during SD partially restored the expression of MTHFR, RAD51, and TOPBP1, suggesting that enhanced folate availability can counteract some of the molecular alterations induced by SD. Collectively, our findings suggest a mechanistic link whereby SD disrupts folate transport and metabolism, potentially compromising DNA repair in sperm and thereby impairing sperm quality and embryonic development. This suggests that targeting folate metabolism may offer a therapeutic strategy to mitigate SD-associated male infertility.