Diacylglycerol kinase theta (DGKθ) plays a pivotal role in regulating adrenocortical steroidogenesis by synthesizing the ligand for the nuclear receptor steroidogenic factor 1 (SF1). In response to activation of the cAMP signaling cascade nuclear DGK activity is rapidly increased, facilitating PA-mediated, SF1-dependent transcription of genes required for cortisol and dehydroepiandrosterone (DHEA) biosynthesis. Based on our previous work identifying DGKθ as the enzyme that produces the agonist for SF1, we generated a tetracycline-inducible H295R stable cell line to express a short hairpin RNA (shRNA) against DGKθ and characterized the effect of silencing DGKθ on adrenocortical gene expression. Genome-wide DNA microarray analysis revealed that silencing DGKθ expression alters the expression of multiple genes, including steroidogenic genes, nuclear receptors and genes involved in sphingolipid, phospholipid and cholesterol metabolism. Interestingly, the expression of sterol regulatory element binding proteins (SREBPs) was also suppressed. Consistent with the suppression of SREBPs, we observed a down-regulation of multiple SREBP target genes, including 3-hydroxy-3-methylglutary coenzyme A reductase (HMG-CoA red) and CYP51, concomitant with a decrease in cellular cholesterol. DGKθ knockdown cells exhibited a reduced capacity to metabolize PA, with a down-regulation of lipin and phospholipase D (PLD) isoforms. In contrast, suppression of DGKθ increased the expression of several genes in the sphingolipid metabolic pathway, including acid ceramidase (ASAH1) and sphingosine kinases (SPHK). In summary, these data demonstrate that DGKθ plays an important role in steroid hormone production in human adrenocortical cells.