Melanin synthesized by epidermal melanocytes protects the skin against UVR-induced DNA damage and skin cancer. Exposure to UVR increases the synthesis of the photoprotective eumelanin on activation of MC1R, a melanoma susceptibility gene. We studied the expression of MC1R under UVR and -MSH stimulation in skin of different ethnic origins and in melanocytes of various pigmentary levels. This study identifies and characterizes a novel MC1R isoform (MC1R350) generated by alternative splicing of the classically known MC1R (MC1R317). We demonstrate that the melanin content of melanocytes shows a significant positive correlation with MC1R317 levels but correlates inversely with the amount of MC1R350, suggesting that this latter isoform could act as a negative regulator of melanin synthesis. We confirmed that hypothesis by showing that while MC1R317 signaling significantly increases the expression of MITF and tyrosinase, two key factors in the melanin synthesis pathway, MC1R350 dramatically hampers their expression. In the skin, we show that UVR does not increase MC1R350 expression but does significantly increase MC1R317. Taken together, our results strongly suggest that MC1R350 acts as a negative regulator of skin pigmentation and demonstrate for the first time that MC1R isoform-specific expression is closely related to skin pigmentation and photoprotection.--Rouzaud, F., Costin, G-E., Yamaguchi, Y., Valencia, J. C., Berens, W. F., Chen, K. G., Hoashi, T., Böhm, M., Abdel-Malek, Z. A., Hearing, V. J. Regulation of constitutive and UVR-induced skin pigmentation by melanocortin 1 receptor isoforms.