Orexins mediate a variety of physiological processes including feeding behaviour, the circadian pathway and cortisol secretion. Steroidogenesis is regulated by a variety of neuropeptides and one of the key rate-limiting steps is cholesterol transport across the mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein. StAR expression can be regulated through several different signalling pathways. Despite the clear link between orexins and steroid production, the actions of the orexin family of hormones on steroid biosynthesis are not fully understood. We present data showing that 100nM both ORA and ORB for 4 or 24h significantly up-regulate StAR, in H295R pluripotent adrenocortical cells. We present the dose-dependent and time-dependent characteristics of StAR up-regulation at the protein level, showing significant increases after 4h at a relatively low agonist concentration (1nM). We have provided a key analysis of the precise G-protein coupled signalling pathways required for the up-regulation of StAR protein in response to ORA and ORB. This has involved dominant negative G-protein analysis and the direct inhibition of the PKA, PKC, ERK1/2 and p38 pathways. This shows a fundamental role for multiple G-protein coupled and MAPK-mediated signalling pathways leading to StAR expression. Antagonist analysis also showed that orexin effects on StAR were primarily (but not exclusively) acting through the OX1R. This is the first study linking orexin action on StAR expression and comprehensively describes the signalling pathways involved in regulating the complexity of hormone biosynthesis.