Gossypin is a flavone that was originally isolated from Hibiscus vitifolius and has traditionally been used for the treatment of diabetes, jaundice, and inflammation. Recently, gossypin was found to have potent anticancer properties; however, its effect on human gliomas still remain unknown. To investigate the potential anticancer effects of gossypin on malignant gliomas and analyze the associated molecular mechanisms, we treated human glioma U251 cells with gossypin. Our study showed that the treatment of U251 cells with gossypin inhibited cell proliferation in a dose- and time-dependent manner and was observed to be minimally toxic to normal human astrocytes. Gossypin's effect on cell cycle distribution was observed, and we found that it induced G2/M-phase arrest in U251 cells. An analysis of cell-cycle regulatory proteins indicated that the arresting effect of gossypin on the cell cycle at G2/M phase was involved in the phosphorylation of cell division cycle 25C (Cdc25C) tyrosine phosphatase via the activation of checkpoint kinase 1 (Chk1). These findings indicate that gossypin is a potential treatment of gliomas because of gossypin's potential to regulate the proliferation of U251 cells via the cell-cycle regulatory proteins Chk1 and Cdc25C.