BACKGROUND: Melanocytes protect the body from ultraviolet radiation by synthesizing melanin. Tyrosinase, a key enzyme in melanin production, accumulates in the endoplasmic reticulum (ER) during melanin synthesis, potentially causing ER stress. However, regulating ER function for melanin synthesis has been less studied than controlling Tyrosinase activity. OBJECTIVE: This study investigates the regulatory mechanisms of melanin production, focusing on ER stress and the ER stress-induced response. METHODS: B16 mouse melanoma cells induced to undergo melanogenesis were treated with unfolded protein response (UPR) inhibitors or chemical chaperones, and their effects on melanogenesis were analyzed. RESULTS: During melanogenesis in B16 cells stimulated by alpha-melanocyte-stimulating hormone (α-MSH), ER stress and UPR activation occurred, accompanied by increased Tyrosinase protein. Reducing IRE1 and ATF6 branch activity lowered melanin levels, while chemical chaperone treatment restored melanin production and increased Tyrosinase levels. CONCLUSION: UPR activation, linked to elevated Tyrosinase levels, influences melanin production during melanogenesis. Modulating UPR can regulate melanin synthesis and provides a potential new approach for treating pigmentation disorders.