Although sunitinib possesses significant clinical effects on imatinib-resistant gastrointestinal stromal tumors (GISTs), the individuals with GIST eventually become resistant to treatment with this tyrosine kinase inhibitor. The mechanism of resistance to sunitinib is still under investigation. To address this issue, we have established sunitinib-resistant GIST-T1 sublines (designated as GIST-T1R) by culturing cells with increasing concentrations of sunitinib. GIST-T1R cells were also resistant to imatinib-mediated growth inhibition. Examination of intracellular signaling found that Akt/ mammalian target of rapamycin (mTOR) signaling remained activated in GIST-T1R but not in parental GIST-T1 cells, after exposure of these cells to sunitinib, as measured by immunoblotting. Further study found that the phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene was silenced by methylation of the promoter region of the gene. Notably, forced-expression of PTEN in GIST-T1R cells negatively regulated the Akt/mTOR pathways and sensitized these cells to sunitinib-mediated growth arrest and apoptosis. Taken together, epigenetic silence of PTEN might be one of the mechanisms which cause drug-resistance in individuals with GIST after exposure to tyrosine kinase inhibitors. Blockade of the PI3K/Akt signaling with the specific inhibitors could be useful in such a case.