Scavenger receptor class B, type I (SR-BI) binds HDL and mediates selective delivery of cholesteryl esters (CEs) to the liver, adrenals, and gonads for product formation (bile acids and steroids). Because relatively little is known about SR-BI posttranslational regulation in steroidogenic cells, we examined the roles of Na(+)/H(+) exchanger regulatory factors (NHERFs) in regulating SR-BI expression, SR-BI-mediated selective CE uptake, and steroidogenesis. NHERF1 and NHERF2 mRNA and protein are expressed at varying levels in model steroidogenic cell lines and the adrenal, with only low expression of PDZK1 (NHERF3) and NHERF4. Dibutyryl cyclic AMP decreased NHERF1 and NHERF2 and increased SR-BI mRNA expression in primary rat granulosa cells and MLTC-1 cells, whereas ACTH had no effect on NHERF1 and NHERF2 mRNA levels but decreased their protein levels in rat adrenals. Co-immunoprecipitation, colocalization, bimolecular fluorescence complementation, and mutational analysis indicated that SR-BI associates with NHERF1 and NHERF2. NHERF1 and NHERF2 down-regulated SR-BI protein expression through inhibition of its de novo synthesis. NHERF1 and NHERF2 also inhibited SR-BI-mediated selective CE transport and steroidogenesis, which were markedly attenuated by partial deletions of the PDZ1 or PDZ2 domain of NHERF1, the PDZ2 domain of NHERF2, or the MERM domains of NHERF1/2 or by gene silencing of NHERF1/2. Moreover, an intact COOH-terminal PDZ recognition motif (EAKL) in SR-BI is needed. Transient transfection of hepatic cell lines with NHERF1 or NHERF2 caused a significant reduction in endogenous protein levels of SR-BI. Collectively, these data establish NHERF1 and NHERF2 as SR-BI protein binding partners that play a negative role in the regulation of SR-BI expression, selective CE transport, and steroidogenesis.