Steroidogenic acute regulatory protein (StAR)/StarD1, a 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. Here, we examined the role of oxidant-sensitive p38 MAPKs in the regulation of StAR gene transcription using model steroidogenic cell lines. Our data show 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 over expression of a dominant-negative (dn) form of p38 MAPKα or p38 MAPKβ construct. Studies with rat StAR-promoter demonstrated that over expression of p38 MAPKα-wt, β or γ significantly reduced both basal and cAMP-sensitive promoter activity. In contrast, overexpression of p38 MAPKα-dn, β or γ enhanced the StAR promoter activity under basal conditions and in response to cAMP stimulation. Use of various constitutively active and dominant negative constructs and designer knockout cell lines demonstrated that MKK3 and MKK6, the upstream activators of p38 MAPKs, play a role in p38 MAPKα mediated inhibition of StAR promoter activity. In addition, our studies raised the possibility of CREB as a potential target of the p38 MAPK inhibitory effect on StAR promoter activity. Collectively, these data provide novel mechanistic information by which oxidant-sensitive p38 MAPKs, particularly p38 MAPKα, contribute to the negative regulation of StAR gene expression and inhibit steroidogenesis.