Objective To establish a mouse model bearing orthotopic temozolomide (TMZ)-resistant glioma that mimics the development of drug resistance in gliomas in vivo. Methods Seventy-eight adult C57BL/6 mice were randomly divided into 6 groups (n=13), including 3 TMZ induced groups with low, medium and high doses (5, 25, and 50 mg/kg, respectively) and 3 control groups. In each group, 5 mice were used for evaluating tumor size, 5 for observing survival, and 3 for collecting tumor tissues for primary cell culture. In low-dose TMZ induced group, 3 mice bearing orthotopic murine glioma GL261 cell xenografts received intraperitoneal injections of 5 mg/kg TMZ for 5 days followed by a 10-day washout period before collecting glioma tissues. Tumor cell suspensions were prepared and injected in the mice in the medium-dose group, which were treated with the same protocol but with an increased TMZ dose, and the tumor cells harvested from 3 mice were injected in the high-dose group. The mice bearing GL261 cell xenografts in the 3 control groups received no treatment or were injected with medium- or high-dose TMZ. Cell colony forming assay was used to assess TMZ resistance of each generation of the tumor cells; CCK8 assay was used to determine drug resistance index of the cells. Results The mouse models bearing TMZ resistant glioma was successfully established. The cells from the high-dose induced group showed a significantly higher colony- forming rate than those from the high-dose control group (P<0.05), and had a drug resistance 4.25 times higher than that of the cells from untreated control group. High-dose TMZ significantly reduced the tumor volume in the control group (P<0.05) but not in the high-dose induced group (P<0.01). The survival time of the tumor-bearing mice was significantly shortened in the high-dose induced group (P=0.0018). Conclusion Progressive increase of TMZ doses in mice bearing orthotopic gliomas can effectively induce TMZ resistance of the gliomas.