Vinblastine (VELBE) is one of the first chemotherapeutic agents used in the treatment of malignancies. To explore the effectiveness of various treatment regimens, bolus versus 24-hour continuous VELBE treatment was tested on an EAT tumor model in mice. Continuous VELBE infusion was simulated by splitting the bolus VELBE dose into 4 fractions, injected at 8-hour intervals. A comparison of antitumor effectiveness between bolus and split dose VELBE treatment was determined by three assays: cell survival, tumor growth delay and animal survival. The cell survival curves of both bolus and split dose VELBE treatments indicated a biphasic response with an initial fast reduction in cell survival followed by a plateau. However, split dose treatment was significantly more effective than bolus treatment at all doses tested (p < 0.001). Tumor growth delay of the split dose VELBE treatment was 6.9 days and of the bolus VELBE treatment 3.0 days, indicating that the split dose treatment is approximately 2-times more effective (p < 0.001). Median survival time of mice treated with split VELBE dose (24.0 days) was significantly longer compared to that of mice treated with bolus VELBE dose (16.5 days) (p < 0.001). The median survival time of control untreated mice (16.0 days) and bolus treated mice did not differ (p = 0.24). Our study shows that at the same VELBE dose the split dose VELBE treatment is more effective than VELBE administered in bolus.