Chemotherapy is the main postsurgical and adjuvant therapy for glioma, and intrinsic or acquired temozolomide (TMZ) resistance may result in poor prognosis. The miR-181 family was discovered to play an important role in regulating biological functions in glioma, and miR-181b is less expressed in human gliomas as a tumor-suppressive miRNA. The aim of this study was to explore the molecular mechanism of miR-181b-5p and its target gene on modulating TMZ chemosensitivity in glioma cells. The enhanced chemosensitivity effect of miR-181b-5p to TMZ in glioma cells U87MG and U251 was detected by MTT method. Dual luciferase reporter assay, quantitative real-time PCR (qRT-PCR) and Western blotting were performed to demonstrate that miR-181b-5p directly targets Bcl-2 to reduce the expression. Transwell and flow cytometry assays showed that combination of miR-181b-5p and TMZ exerted stronger effects on inhibiting U87MG cells proliferation, migration and invasion as well as promoting apoptosis and S phase arrest than miR-181b-5p and TMZ alone. The same tendency was observed in the upregulation of apoptosis-related protein Bax and downregulation of cycle-related proteins CyclinD1 and CDK4. In vivo experiments indicated that miR-181b-5p could enhance the tumor-suppressive effect of TMZ. In conclusion, our findings indicate that upregulation of miR-181b-5p targets Bcl-2 directly and may function as an important modifier to sensitize glioma cells to TMZ.