De novo steroidogenesis is the process by which steroid hormones are synthesized from a common precursor, cholesterol. The first step occurs in mitochondria, where cholesterol is converted to pregnenolone by cholesterol side-chain cleavage enzyme (CYP11A1) on the matrix side of the inner mitochondrial membrane. Steroid hormone production is rapidly stimulated by trophic hormones, necessitating effective mobilization and delivery of cholesterol to the outer mitochondrial membrane (OMM) to support efficient steroidogenesis. My studies redefine the prevailing view that steroidogenic acute regulatory protein (STAR) expression forms the rate-limiting step in steroidogenesis. Using a STAR-knockout MA-10 Leydig cell model, I demonstrate that STAR abundance alone does not define the rate-limiting step in steroidogenesis. Instead, the critical limiting event is the coordinated delivery of cholesterol to the OMM in conjunction with functional STAR activity. These findings establish mitochondrial cholesterol availability — not merely STAR expression — as the principal determinant of de novo steroidogenesis. My studies also examined the potential roles of Oxysterol-Binding Protein-Related Proteins 9 and 11 (ORP9 and ORP11) in cholesterol transfer to the OMM. ORP9 was the most abundant ORP family member expressed in MA-10 Leydig cells, while ORP11 was found to be phosphorylated during steroidogenesis. Both proteins function at membrane contact sites between organelles, and ORP9 forms a heterodimer with ORP11, suggesting a coordinated role in intracellular lipid transport. However, CRISPR/Cas9-mediated deletion of ORP9 or ORP11 did not abolish steroid hormone production, suggesting that these proteins are not essential for cholesterol transfer to the OMM. My studies further demonstrate that the plasma membrane (PM) serves as a critical cholesterol conduit during steroidogenesis, whereby cholesterol — regardless of its source — must first be directed to the PM before delivery to the mitochondria. Using complementary pharmacological and genetic approaches, I showed that PM cholesterol is internalized via dynamin- and clathrin-mediated endocytosis in response to trophic stimulation. Internalized cholesterol containing membrane vesicles are subsequently trafficked through microtubule-dependent vesicular pathways for mitochondria delivery. Furthermore, I investigated the roles of motor proteins, including kinesins and cytoplasmic dynein, in facilitating this directional intracellular transport. These findings reshape our understanding of cholesterol mobilization during steroidogenesis and establish mitochondrial cholesterol availability as a regulated process, thereby opening new avenues for identifying the lipid-transfer proteins that mediate this mechanism.