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Background Glioma is the most common primary brain tumor with poor prognosis. Temozolomide has been used with thalidomide to treat gliomas. We investigated the synergistic mechanism of these two drugs in vitro.Methods Human malignant glioma cells U251-MG were cultured and assigned to four groups with different treatments for 3 days: temozolomide group (100 pmol/L), thalidomide group (100 pg/L), temozolomide (100 IJmol/L) plus thalidomide group (100 pg/L) and control group. MTT assay was applied to evaluate the cell viability. Cell cycle was analyzed by flow cytometry. The ultra-structural features of autophagosomes were observed with electron microscope. Acridine orange and monodansylcadavedne were adopted to label autophagosomes and flow cytometry was applied for quantification of autophagosomes. The expression of autophagy-associated protein was detected by Western blotting.Results Proliferation of tumor cell was obviously suppressed by temozolomide with thalidomide treatment than by either drug used alone (P=-0.000 for each day). The combination treatment induced cell cycle arrest at G0/G1 phase.Typical autophagic ultra-structural character was found after the combined treatment. Thalidomide promoted the autophagy induced by temozolomide. The autophagy-associated proteins - microtubule associated protein 1 light chain 3 (MAPILC3) and Beclinl were more significantly up-regulated by the combined treatment than temozolomide used alone (MAP1LC3, P=-0.000; Beclinl, P=-0.004). The expression level of phosphatase and tensin homolog deleted on chromosome ten (PTEN), which promoted autophagy by suppressing PI3K/Akt/mTOR signaling pathway, was elevated by thalidomide (thalidomide group: P=-0.000; combined group: P=0.002).Conclusions Thalidomide enhances the cytotoxicity of temozolomide by promoting the autophagy induced by temozolomide. Contributing to the up-regulation of PTEN by thalidomide, the expression of autophagy associated protein-MAP1LC3 and Beclinl was enhanced, which leads to a reinforced autophagy in the combined treatment of temozolomide and thalidomide in vitro.