In steroidogenic tissues, cholesterol must be transported to the inner mitochondrial membrane to be converted to pregnenolone as the first step of steroidogenesis. Whereas steroidogenic acute regulatory protein has been shown to be responsible for the transport of cholesterol from the outer to the inner mitochondrial membrane, the process of how cholesterol moves to mitochondria from the cytoplasm is not clearly defined. The involvement of the cytoskeleton has been suggested; however, no specific mechanism has been confirmed. In this paper, using genetic ablation of an intermediate filament protein in mice, we present data demonstrating a marked defect in adrenal and ovarian steroidogenesis in the absence of vimentin. Cosyntropin-stimulated corticosterone production is decreased 35 and 50% in male and female Vimentin null (Vim−/−) mice, respectively, whereas progesterone production is decreased 70% in female Vim−/− mice after pregnant mare's serum gonadotropin and human chorionic gonadotropin stimulation, but no abnormalities in human chorionic gonadotropin-stimulated testosterone production is observed in male Vim−/− mice. These defects in steroid production are also seen in isolated adrenal and granulosa cells in vitro. Further studies show a defect in the movement of cholesterol from the cytosol to mitochondria in Vim−/− cells. Because the mobilization of cholesterol from lipid droplets and its transport to mitochondria is a preferred pathway for the initiation of steroid production in the adrenal and ovary but not the testis and vimentin is a droplet-associated protein, our results suggest that vimentin is involved in the movement of cholesterol from its storage in lipid droplets to mitochondria for steroidogenesis.