Objective: Pigment epithelium-derived factor (PEDF) plays a crucial role in various biological functions such as anti-angiogenesis, neuroprotection, and tumor suppression. Despite its importance, the expression and distribution of PEDF in normal mammalian tissues remain inadequately understood. This study aims to elucidate the expression levels and localization of PEDF across different tissues in healthy sheep, providing insights that may benefit regenerative medicine in sports. Methods: Three healthy sheep were utilized to assess the PEDF expression in diverse tissues including the heart, liver, spleen, lungs, kidneys, cerebrum, cerebellum, caput epididymides, corpora epididymides, cauda epididymides, and testes. Following euthanasia, these tissues were harvested for analysis. The expression and localization of PEDF were examined through quantitative real-time PCR (qRT-PCR), western blotting, and immunohistochemistry. Results: PEDF was variably expressed in all examined tissues, displaying a consistent trend at both mRNA and protein levels. Visceral tissues showed significantly higher PEDF expression in the lungs compared to the heart, liver, spleen, and kidneys (p < 0.01). In brain tissues, PEDF levels were notably higher in the cerebrum than in the cerebellum. Among gonadal tissues, the highest expression was observed in the caput epididymis, with similar levels in the corpora epididymides, cauda epididymides, and testes. Immunohistochemical staining confirmed PEDF presence in the connective tissue between cardiomyocytes, visceral tissue cell cytoplasm, brain tissue nerve fibers, and the cytoplasm of testicular and epididymal epithelial cells. Conclusion: The widespread expression of PEDF mRNA and protein across sheep tissues highlights its potential multifunctional role. The variation in expression levels and locations suggests that PEDF's biological functions are closely linked to its distribution. This study lays the groundwork for future investigations into the therapeutic applications of PEDF, particularly in the field of sports medicine, where its regenerative and protective properties could be harnessed to improve athlete recovery and performance.