In males, androgens are mainly produced by Leydig cells from the testis. A critical and highly regulated step of steroidogenesis involves the importation of cholesterol within the mitochondria by the steroidogenic acute regulatory (STAR) protein. During aging, STAR protein levels in Leydig cells gradually decrease, leading to a reduced entry of cholesterol into mitochondria and lower testosterone production. In addition to preserving its steroidogenic capacity, tumor Leydig cells can also be excellent models for evaluating the mechanisms of action of anticancer agents. In this study, we examined whether polyphenolics having structural similarities to luteolin could promote steroidogenic and cancer-related gene expressions within rat L540 tumor Leydig cells. In this cell model, luteolin activated Star expression and increased progesterone as well as testosterone productions. Interestingly, luteolin decreased gene expression related to cholesterol biosynthesis, possibly inhibiting membrane synthesis and cell proliferation. In addition, increased expression of genes such as Fas, Cdkn1a, Atp7b, and Tp53, as well as increased accumulation of cleaved caspase 3 and PARP, in response to luteolin treatment indicates that apoptosis is being activated. Luteolin also modulated the expression of genes involved in stress response, such as glutathione-S transferases Gsta1 and Gstt2, and the unfolded protein response. Thus, dietary luteolin may be effective in Leydig cell tumor chemoprevention and in maintaining steroidogenesis in aging males.