Objective：This study aims to investigate how phosphorylation regulates the tumor-suppressive activity of Suppressor of fused（Sufu）in medulloblastoma（MB） cells and elucidate the potential mechanism. Methods：The expression and phosphorylation levels of Sufu in MB tissue microarrays were detected by immunohistochemistry. Wild type Sufu or its phosphorylation sites mutated plasmids were transfected into MB cell line DAOY. The cellular distribution of Sufu was detected by ICC and Western blot，and the cell proliferation and apoptosis were detected by CCK8，EdU labeling，and flow cytometry. Besides，the transcriptional activity of Glioma-associated oncogene homologue（Gli） at downstream of Hedgehog（Hh） signaling was analyzed by luciferase reporter assay，and the protein level of ornithine decarboxylase（ODC1）was detected by Western blot in transfected cells. Results：Sufu was highly expressed and phosphorylated in MB tissues，compared to normal cerebellum. Phosphorylated Sufu was mainly concentrated in the nucleus. Unphosphorylation dampened the inhibitory effect of Sufu on proliferation and its promotion of apoptosis in DAOY cells. It is known that Gli-mediated transcriptional activation and ODC1-mediated polyamine synthesis can promote MB cell growth at the downstream of Hh signaling. Phosphorylated Sufu inhibited the transcriptional activity of Gli. Unphosphorylated Sufu showed impaired repressor activity and promoted the expression of ODC1. Conclusion：Phosphorylation enhances the inhibitory effect of Sufu on MB cell growth，which may be related to inhibition of Gli-mediated Hh signaling transduction and the metabolism of polyamines.