Nr2e3 is an orphan nuclear receptor expressed specifically by retinal photoreceptor cells. Mutations in Nr2e3 result in syndromes characterized by excess blue cones and loss of rods: enhanced S-cone syndrome (ESCS) in humans and rd7 in mice. Using yeast two-hybrid screens with Nr2e3 as bait, the cone-rod homeo-box protein Crx was identified as an interacting partner of Nr2e3. Immunoprecipitation assays confirmed this Nr2e3-Crx interaction and identified the DNA-binding domain of each protein as the interaction motif. Immunohistochemistry demonstrated that Crx and Nr2e3 are co-expressed by rod photoreceptors and their precursors. Chromatin immunoprecipitation assays on mouse retina demonstrated that Nr2e3 and Crx co-occupy the promoter/enhancer region of several rod and cone genes in the rod photoreceptor cells. The promoter/enhancer occupancy of Nr2e3 is Crx-dependent, suggesting that Nr2e3 is associated with photoreceptor gene targets by interacting with Crx. Transient transfection assays in HEK293 cells demonstrated that Nr2e3 enhances rhodopsin, but represses S- or M-cone opsin transcription when interacting with Crx. Quantitative real-time RT-PCR analysis on postnatal day 28 (P28) retina of the rd7 mouse, which lacks Nr2e3 protein, revealed an up-regulation of cone genes, but down-regulation of rod genes. Several mutant forms of human Nr2e3 identified from ESCS patients showed defects in interacting with Crx and/or in transcriptional regulatory function. Altogether, our findings suggest that Nr2e3 is a dual-function transcriptional regulator that acts in concert with Crx to promote and maintain the function of rod photoreceptor.