This study was designed to evaluate the protective effect of melatonin against in vitro hemolysis induced by fluoride (F) in human red blood cells. Venous blood samples from twenty healthy, well-nourished male donors, age 20–25, residing in a nonfluorotic area of Ahmedabad, India, were collected for the preparation of red blood cell (RBC) suspensions in normal saline. When 2.0 mL of the RBC suspensions were treated for 4 hr at 37oC with 0.05–0.5 mL of 4 mg NaF/mL in normal saline (50– 500 µg NaF/mL in a final normal saline volume of 4.0 mL), they exhibited a significant dose-dependent increase in hemolysis. Addition of melatonin (5 µg/mL and 10 µg/ mL) to the final volume caused a significant reduction in F-induced hemolysis with maximum amelioration occurring at 10 µg/mL. Melatonin therefore can exert a significant protective action against F-induced hemolysis in vitro.