Increasing evidence indicates that microRNAs (miRNAs) participate in the regulation of chemoresistance in a variety of cancers including glioma. However, the molecular mechanism underlying the development of chemoresistance in glioma is not well understood. The aim of the present study was to explore the role of miRNAs in the chemosensitivity of glioma cells and the underlying mechanism. By microarray and qRT-PCR, we observed significant down-regulation of microRNA-302c (miR-302c) in the temozolomide (TMZ)-resistant human glioma tissues/cells. The low expression of miR-302c was closely associated with poor prognosis and chemotherapy resistant in patients. miR-302c up-regulation re-sensitized U251MG-TMZ cells and LN229-TMZ cells to TMZ treatment, as evidenced by inhibition of the cell viability, cell migration, and invasion capacity, and promotion of the apoptosis after TMZ treatment. Furthermore, P-glycoprotein (P-gp) was identified as a functional target of miR-302c and this was validated using a luciferase reporter assay. In addition, P-gp was found to be highly expressed in U251MG-TMZ cells and there was an inverse correlation between P-gp and miR-302c expression levels in clinical glioma specimens. Most importantly, we further confirmed that overexpression of P-gp reversed the enhanced TMZ-sensitivity induced by miR-302c overexpression in U251MG-TMZ and LN229-TMZ cells. Our finding showed that up-regulation of miR-302c enhanced TMZ-sensitivity by targeting P-gp in TMZ-resistant human glioma cells, which suggests that miR-302c would be potential therapeutic targets for chemotherapy-resistant glioma patients.