Retinoic acid-related orphan receptors (RORs) and the basic helix-loop-helix-PAS transcription factor Npas2 have been implicated in the control of circadian rhythm. In this study, we demonstrate that RORγ directly regulates Npas2 expression in vivo. Although the rhythmicity of Npas2 mRNA expression was maintained in RORγ-/- mice, the peak level of expression was significantly reduced in several tissues, while loss of RORα had little effect. Inversely, overexpression of RORγ in hepatoma Hepa1-6 cells greatly induced the expression of Npas2. RORγ-activated Npas2 transcription directly by binding two ROREs in its proximal promoter. ChIP analysis demonstrated that RORγ was recruited to this promoter in the liver of wild-type mice, but not RORγ-deficient mice. Activation of Npas2 correlated positively with chromatin accessibility and level of H3K9 acetylation. The activation of Npas2 by RORγ was repressed by co-expression with Rev-Erbα or addition of the ROR inverse agonist T0901317. Npas2 expression was also significantly enhanced during brown adipose differentiation and that this induction was greatly suppressed in adipose cells lacking RORγ. Our results indicate that RORγ and Rev-Erbα are part of a feed-back loop that regulates the circadian expression of Npas2 suggesting a regulatory role for these receptors in Npas2-dependent physiological processes. © 2011 The Author(s).