Three new 7-0-substituted deacetamidothiocolchicine derivatives have been evaluated for their antitumor activity against various human tumor cell lines, some of which express the multidrug resistance (MDR) phenotype, for their impact on the cell cycle and their binding to tubulin. Colchicine and thiocolchicine were used as reference compounds. Thiocolchicine was the most active agent on MDR-negative cells in terms of growth inhibition, whereas for multidrug-resistant cells, thiocolchicone was the most active compound (IC50 = 14 nM). As indicated by statistical analysis, a perfect agreement for the potency order (IC50 values) of the compounds between all the MDR-negative cancer cells (k = 1.00), a poor agreement between MDR-positive and MDR-negative cancer lines, and a moderate agreement (k = 0.50) between the two resistant cancer cells MCF-7 ADRr and CEM VBL were observed. To gain further insight into the mechanism of the antitumor activity of colchicinoids, the most active compounds, colchicone and thiocolchicone, were selected to evaluate their effect on cell cycle, apoptosis, and tubulin interaction. The highest recruitment activity into the G21/M phase of the cell cycle was detected in thiocolchicone-treated breast cancer cells. Interestingly, after 72 h of culture, when the cell cycle block subsided, a consistent amount of DNA fragmentation, a hallmark of apoptosis, was evident. Morphological analysis of MCF-7 ADRr cells confirmed this hypothesis and revealed that thiocolchicone was able to induce apoptosis in this MDR-bearing model. We also demonstrated, using flow cytometry, that thiocolchicone interacts with alpha- and beta-tubulin, thereby affecting the expression of both subunits.