In this study, we identify Prospero-related homeobox 1 (Prox1) as a novel co-repressor of the retinoic acid-related orphan receptors, RORα and RORγ. Prox1 interacts directly with RORγ and RORα and negatively regulates their transcriptional activity. The AF2 domain of RORs is essential for the interaction, whereas Prox1 interacts with RORs through either its 28 amino acids N-terminal region or its C-terminal prospero-like domain. RORγ antagonists stabilize the interaction between RORγ and Prox1. The homeodomain and the interaction through the prospero-like domain of Prox1 are critical for its repression of ROR transcriptional activity. Chromatin immunoprecipitation analysis demonstrated that in liver, Prox1 is recruited to the ROR response element sites of the clock genes, brain and muscle Arnt-like protein 1 (Bmal1), neuronal PAS domain protein 2 (Npas2) and cryptochrome 1 (Cry1), as part of the same complex as RORs. Knockdown of Prox1 by siRNAs in human hepatoma Huh-7 cells increased the expression of RORγ and several ROR-target genes, along with increased histone acetylation at these ROR response element sites. Chromatin immunoprecipitation sequencing analysis suggests that Prox1 is a potential ROR target gene in liver, which is supported by the regulation of the rhythmic expression of Prox1 by RORγ. Our data suggest that Prox1 is part of a feedback loop that negatively regulates the transcriptional control of clock and metabolic networks by RORs.