Objective To investigate the effect of melatonin on the proliferation, function and p38 MAPK protein expression of pancreatic β cells. MethodIns-1 cells were used as the research object. Six treatment groups and one control group were divided according to the concentration of melatonin to observe the dose-effect relationship of melatonin on the proliferation and function of INS-1 cells. The proliferation rate was detected by CCK-8 method, and the glucose-stimulated insulin secretion (GSIS) function was detected by ELISA. Four groups were divided to observe the effect of melatonin on the expression of p38 MAPK protein, and the expression levels of p38 MAPK and p-p38 MAPK protein were detected by Western blot. Results Compared with the control group, the proliferation rate of each treatment group decreased significantly, and the cell proliferation rate first increased and then decreased with the increase of melatonin concentration, and the proliferation rate of the 10 nmol/L melatonin group was the highest. Compared with the control group, the insulin concentrations of the 1 nmol/L, 5 nmol/L, 25 nmol/L, 50 nmol/L and 100 nmol/L melatonin groups were significantly decreased, and the differences were statistically significant (all P<0.05). The increase of melatonin concentration first increased and then decreased, with the highest in the 10 nmol/L melatonin group. Compared with the control group, both the melatonin group and the SB203580 group could significantly reduce the expression levels of p38 MAPK and p-p38 MAPK proteins (both P<0.05); compared with the melatonin group or the SB203580 group, the expression levels of p38 MAPK and p-p38 MAPK proteins in the combined group were significantly decreased (both P<0.05). Conclusion Melatonin has an inverted U-shaped damage on islet β cells, and 10 nmol/L melatonin has the least damage; melatonin inhibits the p38 MAPK signaling pathway of islet β cells.