Previous studies from this and other laboratories have shown that the novel microtubule poison, JG-03-14, which binds to the colchicine binding site of tubulin, has the capacity to promote both autophagy and apoptosis in breast tumor cells, as well as interfering with endothelial cell function and potentially disrupting tumor vasculature. The current work was designed to investigate the interaction between JG-03-14 and cell culture models of colon carcinoma and melanoma, specifically HCT116 human colon carcinoma cells and B16F10 murine melanoma cells. In both cases, JG-03-14 promoted death in the bulk of the treated population. FACS analysis, DAPI and TUNEL staining indicated that only a small fraction of the cell population was undergoing apoptosis; furthermore, there was no evidence of mitotic catastrophe (micronuclei in bi-nucleated cells). Staining with acridine orange and monodansylcadaverine as well as electron microscopy demonstrated the formation of autophagic vesicles, consistent with the cells undergoing extensive autophagy. Cell cycle analysis indicated that cells had arrested in the G2/M stage, with evidence of a hyperdiploid population. Residual surviving cells appeared to be in a state of senescence; furthermore, the senescent cells failed to recover proliferative capacity, indicating that the cells were reproductively dead. Toxicity studies in cardiomyocytes with comparisons to combretastatin and taxol indicated that JG-03-14 was the less toxic of the microtubule poisons. In summary, our studies indicate that JG-03-14 induces autophagic and reproductive cell death in HCT116 colon carcinoma cells and B16F10 murine melanoma cells with limited toxicity to the normal cells that are generally susceptible to taxol and combretastatin. The possibility of alternative mode(s) of cell death (autophagy and irreversible senescence) induced by JG-03-14 makes it a potentially useful candidate as a chemotherapeutic drug that could be used to treat cancers resistant to apoptosis. The relative lack of toxicity of JG-03-14 provides additional support for its potential use in the treatment of malignancies.