Smtnl2 is an epithelial Smoothelin that binds to actin filaments and is crucial for epithelial morphogenesis. We examined the role of Smtnl2 in Sertoli cells, which undergo dynamic cytoskeleton reorganization to phagocytose apoptotic germ cells, a process known as efferocytosis. We observed Smtnl2 expression in primary mouse Sertoli cells and the 15P1 Sertoli cell line. Smtnl2 expression increased in 15P1 cells committing efferocytosis. Smtnl2-deficient Sertoli cells exhibited defective ability to engulf apoptotic germ cells and importantly, the phenomenon occurred in the setting of an unaffected maturation of phagosome. We demonstrated that Smtnl2 regulates the engulfment process through the function of branched actin nucleation protein ARP3, an actin assembly dictator. Intriguingly, a shift in glucose metabolism that restricts lactate production in Sertoli cells was induced upon Smtnl2 depletion, leading to the activation of downstream AMPK and AKT signaling. Using an in vivo RNAi approach, we found that silencing of Smtnl2 in testis triggers an obvious disruption in cytoskeleton architecture and blood-testis barrier integrity across seminiferous epithelium, causing the detachment of massive germ cells from their nest, as evidenced by their exfoliation into the lumen. Overall, our study identifies Smtnl2 as a determinant for Sertoli cells' functioning in supporting spermatogenesis. [Display omitted] • Smtnl2 is an actin filament-binding protein required for mouse Sertoli cells function. • RNA silencing of Smtnl2 in cultured Sertoli cells impairs efferocytosis and lactate biosynthesis. • Smtnl2 orchestrates actin assembly by recruitment of ARP3. • RNA inhibition of Smtnl2 in mouse testis disrupts spermatogenesis and the blood-testis barrier. [ABSTRACT FROM AUTHOR]