Purpose: T cell immunoglobulin-3 (Tim-3) as a negative regulator of anti-tumor immunity has been widely understood. However, the mechanism that Tim-3 depresses anti-tumor are unclear in drug-resistance glioma cells. The present research corroborates Tim-3 expression in drug-resistance glioma cells. Materials and methods: Two glioma sublines (U87 and U251) which had been continually cultured were retested for the study. Cell proliferation activity, cell viability in early and late stage, the expression of the Tim-3 RNA, were detected by CCK-8 and real-time PCR experiments. Enhancement of sensitivity of glioma cells to chemotherapeutic agent was tested after inhibiting Tim-3 expression by ADV-antisense Tim-3. Results: U87 and U251 glioma cells were killed by various doses of temozolomide (TMZ). The CCK-8 experiment showed that the low, middle and high dose of TMZ could advance the cell apoptosis. With the increase of TMZ concentration, the ratio of apoptosis cells accordingly augmented. Expression of Tim-3 was higher in the high dose groups than the lower and middle, but BAT3 decreased. The difference between the two groups was statistically significant (P<0.05). The killing effect increased after interfering Tim-3 expression by ADV-antisense Tim-3. Tim-3 expression was associated with drug resistant glioma cells. Conclusion: Tim-3 expression in the drug-resistant glioma cells leads to resistance to therapeutic reagents. Significantly, down-regulating the expression of Tim-3 improved the potential of TMZ treatment. Tim-3 expression may play an antiapoptotic functional role in drug-resistance glioma cells by protecting cancer cells.