This study aimed to elucidate how the epididymis of high-altitude Tibetan sheep regulates sperm maturation. Epididymal caput, corpus, and cauda tissues were collected from six Tibetan sheep in Haiyan County, Qinghai Province, and subjected to DIA proteomic analysis to identify key signaling pathways and core molecules involved in sperm maturation. Ultimately, we sought to clarify the molecular mechanisms by which the Tibetan sheep epididymis modulates sperm maturation under long-term high-altitude adaptation and evolution.