In this study, the effects of pharmacological concentrations of melatonin (1 μM-1 mM) on human pancreatic stellate cells (HPSCs) have been examined. Cell type-specific markers and expression of melatonin receptors were analyzed by western blot analysis. Changes in intracellular free Ca concentration were followed by fluorimetric analysis of fura-2-loaded cells. Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were determined by fluorescence techniques. Production of reactive oxygen species (ROS) was monitored following 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester and MitoSOX™ Red-derived fluorescence. Cell viability was studied using the AlamarBlue test. Cultured cells expressed markers typical of stellate cells. However, cell membrane receptors for melatonin could not be detected. Thapsigargin, bradykinin, or melatonin induced changes in intracellular free Ca concentration. In the presence of the indole, a decrease in the GSH/GSSG ratio was observed that depended on the concentration of melatonin used. Furthermore, the indole evoked a concentration-dependent increase in ROS production in the mitochondria and in the cytosol. Finally, melatonin decreased HPSC viability in a time and concentration-dependent manner. We conclude that melatonin, at pharmacological concentrations, induces changes in the oxidative state of HPSC. This might regulate cellular viability and could not involve specific plasma membrane receptors.