In the human adrenal cortex, ACTH (ACTH) activates steroid hormone biosynthesis by acutely increasing cholesterol delivery to the mitochondrion and chronically increasing the transcription of steroidogenic genes (including CYP17) via a cAMP-dependent pathway. In the present study, we characterized the role of sphingolipids in ACTH-dependent steroidogenesis. H295R human adrenocortical cells were treated with ACTH or dibutyryl cAMP (Bt₂cAMP) and the content of several sphingolipid species quantified by mass spectrometry. Both ACTH and Bt₂cAMP decreased cellular amounts of several sphingolipids, including sphingomyelin, ceramides and sphingosine, while stimulating the activity of sphingosine kinase and increasing the release of sphingosine-1-phosphate (S1P) into the media. S1P increased CYP17 mRNA expression by promoting the cleavage and nuclear localization of sterol regulatory element binding protein 1 (SREBP1). Chromatin immunoprecipitation assays revealed that Bt₂cAMP and S1P increased acetylation of histone H3 and promoted binding of SREBP1 to the -520/-331 region of the CYP17 promoter. In summary, our studies demonstrate a role for sphingolipid metabolism and SREBP1 in ACTH-dependent CYP17 regulation and steroidogenesis.