STAR/StarD1, part of a protein complex, mediates the transport of cholesterol from the outer to inner mitochondrial membrane, which is the rate-limiting step for steroidogenesis, and where steroid hormone synthesis begins. Herein, we examined the role of oxidant-sensitive p38 MAPKs in the regulation of STAR gene transcription, using model steroidogenic cell lines. Our data indicate that oxidant activation of p38 MAPK exhibits a negative regulatory role in the induction of functional expression of STAR, as evidenced by enhanced induction of STAR (mRNA/protein) expression and increased steroidogenesis during pharmacological inhibition of p38 MAPK or in cells with increased transient overexpression of a dominant-negative (dn) form of p38 MAPKalpha or p38 MAPKbeta. Studies with rat Star-promoter demonstrated that overexpression of p38 MAPKalpha-wt, -beta, or -gamma significantly reduced both basal and cAMP-sensitive promoter activity. In contrast, overexpression of p38 MAPKalpha-dn, -beta, or -gamma enhanced the Star promoter activity under basal conditions and in response to cAMP stimulation. Use of various constitutively active and dn constructs and designer knock-out cell lines demonstrated that MKK3 and MKK6, the upstream activators of p38 MAPKs, play a role in p38 MAPKalpha-mediated inhibition of Star promoter activity. In addition, our studies raised the possibility of CREB being a potential target of the p38 MAPK inhibitory effect on Star promoter activity. Collectively, these data provide novel mechanistic information about how oxidant-sensitive p38 MAPKs, particularly p38 MAPKalpha, contribute to the negative regulation of Star gene expression and inhibit steroidogenesis.