The epididymis plays a significant role as a quality control organ for long-term sperm storage, maturation, and fertilizing ability and perform filtration function to eliminate abnormal or residual spermatozoa by phagocytosis. However, the role of autophagy in spermiophagy during sperm storage in turtle epididymis still needs to be studied. In this study, we reported in vivo spermiophagy via the cellular evidence of lysosome engulfment and autophagy within the principal cells during sperm storage in the turtle epididymis. Using immunofluorescence, Lysosome associated membrane protein-1 (LAMP1) and microtubule-associate protein light chain 3 (LC3) showed strong immunosignals within the apical cytoplasm of epididymal epithelia during hibernation than non-hibernation. Co-immunolabeling of LAMP1 and LC3 was strong around the phagocytosed spermatozoa in the epididymal epithelia and protein signaling of LAMP1 and LC3 was confirmed by western blotting. During hibernation, ultrastructure showed epididymal principal cells were involved in spermiophagy and characterized by the membrane’s concentric layers around phagocytosed segments of spermatozoa, degenerative changes in the sperm head and lysosome direct attachment, and with the existence of cellular components related to autophagy (autophagosome, autolysosome). In conclusion, spermiophagy occurs by lysosomal engulfment and autophagic activity within the principal cells of the turtle epididymis during sperm storage.