© 2020 Elsevier Inc.p97/valosin-containing protein (VCP) is expressed in many cells and plays critical functions in a broad range of diverse cellular processes. Because it is expressed in the mouse testes, predominantly in Sertoli cells, and is known to play a critical role in autophagy and apoptosis in different cell types, we set out to investigate its function in autophagosome maturation, apoptosis and cell cycle arrest in a mouse Sertoli cell line. To study the mechanism of p97/VCP action, p97/VCP siRNA and a specific p97/VCP inhibitor, N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ), were used in the mouse 15P1 Sertoli cell line. Loss of p97/VCP activity due to DBeQ exposure and silencing of p97/VCP (siVCP) expression results in autophagosome (LC3 and p62) accumulation in the cytoplasm of Sertoli cells. The coexpression of autophagosomal and lysosomal markers (LAMP1 and LAMP2) was reduced in cells in which p97/VCP expression had been inactivated. To better understand in which step of autophagy p97/VCP functions, the interaction between autophagosomal and autolysosomal markers was studied by coimmunoprecipitation and colocalization experiments. The interaction between autophagosomal markers and lysosomal markers decreased in siVCP-expressing and DBeQ-exposed cells. Moreover, the expression of siVCP and DBeQ exposure caused cytoplasmic vacuolation, induced caspase 3-7-mediated cell death and decreased cell cycle progression in mouse Sertoli cells. Taken together, the results show that p97/VCP is essential for autophagosome maturation and cell survival in mouse Sertoli cells. When these functions are prevented, impaired autophagy and apoptosis may have a detrimental effect on germ cells and cause male infertility.