It has been recently demonstrated that melatonin protects cells from calyculin A (CA)-induced neurofilament hyperphosphorylation. To further explore the mechanism of melatonin, the wild type neuroblast cells (N2awt) were treated with CA or CA and melatonin or CA and vitamin E, and detected the levels of tau phosphorylation, the activities of GSK-3 and PP-2A, as well as the antioxidant activities of melatonin and vitamin E against CA. It has been found that the antioxidant activity of melatonin and its protection on tau hyperphosphorylation at Ser199/202 and Ser396/404 and Ser422 sites are stronger than that of vitamin E, at the same time, melatonin increases the activity of PP-2A and decreases the activity of GSK-3 while vitamin E decreases the activities of PP-2A and GSK-3. These results suggest that melatonin protects neuroblast cells from CA-induced cytotoxicities not only through its antioxidant effect but also through its regulation of the phosphorylation system.