The outer mitochondrial membrane translocator protein (TSPO) binds cholesterol with high affinity and is involved in mediating its delivery into mitochondria, the rate-limiting step in hormone-induced steroidogenesis. Specific ligand binding to TSPO has been shown to initiate steroid formation. However, recent studies of the genetic deletion of Tspo have provided conflicting results. Here, we address and extend previous studies by examining the effects of Tspo-specific mutations on steroid formation in hormone- and cyclic adenosine monophosphate (cAMP)-responsive MA-10 cells, using the CRISPR/Cas9 system. Two mutant subcell lines, nG1 and G2G, each carrying a Tspo exon2-specific genome modification, and two control subcell lines, G1 and HH, each carrying a wild-type Tspo, were produced. In response to dibutyryl cAMP, the nG1 and G2G cells produced progesterone at levels significantly lower than those produced by the corresponding control cells G1 and HH. Neutral lipid homeostasis, which provides free cholesterol for steroid biosynthesis, was altered significantly in the Tspo mutant cells. Interestingly, the mitochondrial membrane potential (ΔΨm) of the Tspo mutant cells was significantly reduced compared with that of the control cells, likely because of TSPO interactions with the voltage-dependent anion channel and tubulin at the outer mitochondrial membrane. Steroidogenic acute regulatory protein (STAR) expression was induced in nG1 cells, suggesting that reduced TSPO affected STAR synthesis and/or processing. Taken together, these results provide further evidence for the critical role of TSPO in steroid biosynthesis and suggest that it may function at least in part via its regulation of ΔΨm and effects on STAR.