Interstitial chemotherapeutic drug infusion can bypass the blood-brain barrier, and provide high regional drug concentrations without systemic exposure. However, toxicity and efficacy for drugs administered via interstitial continuous (i.c.) infusion have not been characterized. In the current study, vincristine (VIN) was infused into the right frontal lobes of healthy Fisher 344 rats at 30, 45, 60, and 120 μg/ml over a period of 7 days at 1 μl/h, using an Alzet osmotic pump to evaluate toxicity. C6 rat glioblastoma cells transduced with a luciferase gene were inoculated into the right frontal lobe of a second group of rats. VIN was administered to tumor bearing rats via i.c. infusion 7 days later and tumor growth was monitored by bioluminescence intensity (BLI) to assess VIN efficacy, intravenous (i.v.) drug administration was used as a comparison drug delivery method. The results suggested that VIN toxicity is dose-dependent. Efficacy studies showed increased BLI, which correlates with histopathological tumor size, in saline-infused and i.v.-treated tumor-bearing rats. These rats survived an average of 28 ± 0.85 days and 33 ± 1.38 days, respectively. Both groups had large tumors at the time of death. Animals treated with VIN via i.c. infusion survived until day 90, the observation endpoint for this study. This was significantly longer than average survival times in the previous two groups. These results demonstrate that VIN via i.c. infusion is effective in reducing C6 glioblastoma tumors and prolonging rodent survival time compared to i.v. injection and suggest that chemotherapeutic drug administration via i.c. infusion may be a promising strategy for treating malignant brain tumors.