<p>Base excision repair (BER) is essential for maintaining genomic stability, with DNA polymerase β (Pol β) serving as a key enzyme. While Pol β dysfunction has been linked to various diseases, its role in male reproductive aging and Leydig cell (LC) homeostasis remains poorly defined. This study aimed to investigate whether aging-induced downregulation of Pol β impairs BER in LCs, leading to DNA damage accumulation, LC loss, and subsequent testicular dysfunction. We generated Pol β heterozygous (Pol β^+/−) mice using CRISPR-Cas9 technology and analyzed testicular phenotype, serum testosterone, bone mineral density (BMD), LC survival, and sperm motility. Young (6–8&#xa0;weeks) and middle-aged (8&#xa0;months) wild-type mice were compared for Pol β expression, BER efficiency, and DNA damage markers (8-OHdG, AP sites, and single-strand breaks). Gain- and loss-of-function experiments were performed in isolated LCs via siRNA knockdown and overexpression. Besides, young mice were treated with 5-fluorouracil (5-FU) to pharmacologically induce BER stress. Pol β^+/− mice exhibited reduced serum testosterone, lower BMD, LC depletion, and impaired sperm motility. Aging significantly decreased Pol β expression and BER efficiency in LCs, accompanied by increased DNA damage. Pol β knockdown in young LCs recapitulated aging-like phenotypes, including elevated DNA damage and apoptosis. Conversely, Pol β overexpression in middle-aged LCs reduced DNA damage and improved cell survival. Pharmacological induction of BER stress with 5-FU in young mice resulted in similar defects in testosterone levels, LC viability, and DNA integrity. Our findings demonstrated that aging downregulates Pol β, impairing BER in LCs and leading to DNA damage accumulation and testicular decline. Restoration of Pol β mitigates these defects, identifying Pol β-mediated BER as a critical regulator of male reproductive aging and a potential therapeutic target.</p> Graphical Abstract <p></p>