This study investigated the preventive effect of heat-killed Lactobacillus plantarum (L. plantarum) on cholestasis-induced male reproductive toxicity in rats. Rats were divided into control normal, sham control, bile duct ligation (BDL) control, and BDL with heat-killed L. plantarum supplementation groups. The effects on sexual hormones, testicular and epididymal histology, sperm parameters, oxidative stress markers, and inflammatory gene expression were evaluated. Compared to the BDL control group, the BDL + heat-killed L. plantarum group showed higher levels of normal sperm, luteinizing hormone, testosterone, total antioxidant capacity, and catalase activity, indicating improved reproductive function. Conversely, markers of oxidative stress, such as total oxidative status, oxidative stress index, and carbonyl protein, were lower in the BDL + heat-killed L. plantarum group. The expression levels of inflammatory genes tumor necrosis factor-alpha and interleukin-6 were reduced, while interleukin-10 gene expression was increased in the BDL + heat-killed L. plantarum group. Histological evaluation confirmed the positive effects of heat-killed L. plantarum intervention on testicular parameters. In conclusion, heat-killed L. plantarum supplementation protects against cholestasis-induced male reproductive dysfunction in rats, as evidenced by improvements in hormonal balance, sperm quality, oxidative stress, and inflammation.