Objective：To investigate the effect of melatonin on proliferation，migration and invasion of two rhabdomyosarcoma cell lines RD and RH30 and its mechanism. Methods：The proliferation，migration and invasion of both groups were detected by MTT，EdU and Transwell，respectively. Western blot was used to detect the expression of Gli1 in the downstream transcription factor of sonic hedgehog （Shh） before and after MT treatment. After inhibition and over-expression of Gli1 in the two groups，cell proliferation，migration and invasion were detected. Results：MT effectively inhibited the proliferation，migration and invasion of RH30 cells，but had no obvious effect on RD cells，and luzindole，the MT receptor inhibitor，did not change the inhibitory effect of MT on RH30 proliferation. The expression of Gli1 in RH30 cells decreased after MT treatment，and the cell proliferation，migration and invasion changed after Gli1 expression was changed. Conclusion：MT can significantly inhibit the proliferation，migration and invasion of RH30 cells，and its mechanism is related to the inhibition of Gli1 expression in RH30 cells by MT.