The pigment epithelium-derived factor (PEDF), a member of the non-inhibitory serpin family, is distributed in mammals and has various biological functions including the inhibition of angiogenesis, neuroprotection and anti-tumor. Currently, the distribution and expression of PEDF in normal physiological mammal remains unclear. In the current study, three health sheep were used to investigate the PEDF expression and localization in different tissues. Animals are euthanized, and the collected specimens included the heart, liver, spleen, lungs, kidneys, cerebrum, cerebellum, caput epididymides, corpora epididymides, cauda epididymides and testes. Expression and localization of PEDF in these tissues were detected by using qRT- PCR, western blotting and immunohistochemical localization, respectively. The PEDF was expressed in different degrees in all tissues and organs of the sheep, with consistent overall trend at mRNA and protein levels. In visceral tissues, the expression of PEDF was higher (p < 0.01) in lung than in heart, liver, spleen and kidney tissues. In brain tissues, the expression of PEDF was higher (p < 0.01) in cerebrum than in cerebellum. In gonadal tissues, the PEDF expression was higher (p < 0.01) in the caput epididymis than in corpora epididymides, cauda epididymides and testes with no difference among these three tissues. Immunohistochemical results showed that the PEDF was expressed in the connective tissue between cardiomyocytes, cytoplasm of visceral tissues cells, in nerve fibers of the brain tissue, and in the cytoplasm of testicular and epididymal epithelial cells, confirming the results of mRNA and protein expression levels. The findings herein indicated that PEDF mRNA and protein are widely expressed in tissues of sheep. The biological function of PEDF is strongly related to its location and expression level, therefore, our study provides a basis for further study of the different biological functions of PEDF in various tissues.