We sought to identify and characterize microRNA (miRNAs) that posttranscriptionally regulate the expression of scavenger receptor class B type I (SR-BI) and SR-BI-linked selective high-density lipoprotein (HDL) cholesteryl ester (CE) transport and steroidogenesis. Four miRNAs (miRNA-125a, miRNA-125b, miRNA-145, and miRNA-455) with a potential to regulate SR-BI were identified in silico and validated by quantitative real-time PCR (qRT-PCR), Western blot analysis, and SR-BI 3' untranslated region (UTR) reporter assays. In vitro treatment of primary rat granulosa cells and MLTC-1 cells with cyclic AMP (cAMP) or in vivo treatment of rat adrenals with adrenocorticotropic hormone (ACTH) decreased the expression of miRNA-125a, miRNA-125b, and miRNA-455 and reciprocally increased SR-BI expression. Using luciferase constructs containing the 3' untranslated region of SR-BI combined with miRNA overexpression and mutagenesis, we have provided evidence that steroidogenic SR-BI is a direct target of miRNA-125a and miRNA-455. Moreover, the transfection of Leydig tumor cells with precursor miRNA 125a (pre-miRNA-125a) or pre-miRNA-455 resulted in the suppression of SR-BI at both the transcript and protein levels and reduced selective HDL CE uptake and HDL-stimulated progesterone production. Transfection of liver Hepa 1-6 cells with pre-miRNA-125a significantly reduced SR-BI expression and its selective transport function. In contrast, overexpression of miRNA-145 did not affect SR-BI expression or selective HDL CE uptake mediated by SR-BI in steroidogenic cell lines. These data suggest that a trophic hormone and cAMP inversely regulate the expression of SR-BI and miRNA-125a and miRNA-455 in steroidogenic tissues/cells and that both miRNA-125a and miRNA-455, by targeting steroidogenic SR-BI, negatively regulate selective HDL CE uptake and HDL CE-supported steroid hormone production.