Color vision requires the expression of opsin photopigments with different wavelength sensitivities in retinal cone photoreceptors. The basic color visual system of mammals is dichromatic, involving differential expression in the cone population of two opsins with sensitivity to short (S, blue) or medium (M, green) wavelengths. However, little is known of the factors that directly activate these opsin genes and thereby contribute to the S or M opsin identity of the cone. We report that the orphan nuclear receptor RORbeta (retinoid-related orphan receptor beta) activates the S opsin gene (Opn1sw) through binding sites upstream of the gene. RORbeta lacks a known physiological ligand and activates the Opn1sw promoter modestly alone but strongly in synergy with the retinal cone-rod homeobox factor (CRX), suggesting a cooperative means of enhancing RORbeta activity. Comparison of wild-type and mutant lacZ reporter transgenes showed that the RORbeta-binding sites in Opn1sw are required for expression in mouse retina. RORbeta-deficient mice fail to induce S opsin appropriately during postnatal cone development. Photoreceptors in these mice also lack outer segments, indicating additional functions for RORbeta in photoreceptor morphological maturation. The results identify Opn1sw as a target gene for RORbeta and suggest a key role for RORbeta in regulating opsin expression in the color visual system.