Zinc deficiency has a huge impact on male reproduction. The zinc transporter (ZnT) family is involved in the maintenance of zinc homeostasis and testosterone synthesis. However, the underlying mechanisms remain to be investigated. Therefore, in this study, we aimed to determine the effect of zinc transporter 4 (ZnT4) on testosterone synthesis in male Kunming mice and mouse Leydig cells. The results of this study showed that compared with the zinc normal diet group (Con group), the zinc-deficient diet group (ZnD group) had decreased zinc content and increased ZnT4 expression in testicular tissues, and decreased serum testosterone levels, suggesting that ZnT4 may be involved in Leydig cell injury resulting from a zinc-deficient diet. Subsequently, mouse Leydig cell line TM3 cells were used to analyze the effect of ZnT4 downregulation on TM3 cell proliferation and apoptosis, on testosterone synthesis, and its underlying mechanisms. Here, we show that knockdown of ZnT4 can induce the accumulation of zinc, inhibit the viability, and induce apoptosis in TM3 cells. In addition, knockdown of ZnT4 downregulated testosterone concentration and expression of testosterone synthesis–related proteins steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase/D5-D4 isomerase (3β-HSD) in TM3 cells, while hCG could rescue their levels. We show that it is ZnT4 that plays a role in testosterone production through a mediated PI3K/Akt/mTOR autophagy pathway, whereas mTORC1 complex inhibitor (Rapa) blocks the decrease in testosterone levels caused by ZnT4 downregulation. In conclusion, the above results indicate that ZnT4 plays an important role in regulating testosterone synthesis.