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Epididymal specific proteins play a crucial role in sperm maturation. Some of the post-translational modified proteins are transported from the caput to the cauda of the epididymis through exosomes which regulate the function of sperm in cauda epididymis. Rat beta-galactosidase-1-like protein 4 (GLB1L4) expressed specifically in the caput epididymis, localizes on the sperm; however, the regulatory ways in which GLB1L4 protein interacts with sperm to maintain sperm function are unclear. In this study, knockdown of rat GLB1L4 could inhibit in vitro capacitation of sperm in cauda epididymis and reduce the fertility of the male rats by injection of special lentivirus-shRNA into caput epididymis. Moreover, a considerable proportion of GLB1L4 proteins from rat caput epididymis were loaded on exosomes. The exosomes loaded GLB1L4 from in vitro primary rat caput epididymal epithelial cells could bind with spermatozoa in cauda epididymis. Further, the palmitoylation status of cysteine residues at the 12th and 15th sites of the protein molecule could significantly affect cellular localization of GLB1L4 protein. It was identified that most of GLB1L4 was palmitoylated in the presence of exosomes from primary caput epididymal cells and the level of palmitoylated GLB1L4 in the exosomes could be inhibited by 2-bromopalmitate (2-BP). These results suggested that the palmitoylated GLB1L4 from rat caput epididymis could be transported to the cauda epididymis to regulate the sperm function by exosomes. [ABSTRACT FROM AUTHOR]