Non-neuronal dopamine production has not been understood despite dopamine function in non-neuronal tissues. Tyrosinase is a non-neuronal enzyme which converts tyrosine to L-DOPA (l-3,4-dihydroxyphenylalanine) and L-DOPA to l-dopaquinone for further melanin production. Since L-DOPA is a dopamine precursor in neurons, we hypothesized that tyrosinase-derived L-DOPA could alternatively be converted to dopamine. Therefore, this study investigated whether copper supplementation enhanced pigmentation and induced dopamine production via tyrosinase activation in APRE19 cells. Copper is known as a tyrosinase cofactor. In two separate experiments, we cultured ARPE19 in 1% FBS/DMEM with/without 10 μM copper sulfate for approximately 100 days. After 40-50 days, slight pigmentation with copper treatment was confirmed in the cell pellets, while no pigmentation was observed in the non-copper control. After 90-100 days, the pigmentation in the copper treatment group was obvious, while minimal pigmentation was observed in the non-copper control. Dopamine was not detected at 40-50 days in either group, while it was detected after 90-100 days of culture only in the copper-treated group. Tyrosinase mRNA expression was confirmed in both groups at a similar level, while tyrosinase protein expression was significantly higher in the copper treatment group than in the non-copper control. Thus, we determined that copper supplementation efficiently enhances pigmentation and induces dopamine production in long-term culture ARPE19, likely due to increased tyrosinase protein expression and activity. This is the first report showing the significance of copper in non-neuronal dopamine production of RPE cells, which suggests that tyrosinase may be responsible for non-neuronal dopamine production.