Microtubules, dynamic cytoskeletal polymers whose structural subunit is tubulin, represent a well-established target for anticancer drug discovery. The development of small-molecule inhibitors of tubulin polymerization continues to be a productive strategy for identifying new chemotherapeutic agents. Based on structural analysis of known colchicine-binding site inhibitors (CBSIs) and available tubulin-ligand models, a novel series of 6-aryl-1-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-c]pyridine derivatives were designed and synthesized as potential CBSIs. Among these, compound 15u showed the most potent antiproliferative activity, with IC50 values of 0.090-0.12 μM against three human cancer cell lines (Huh7, HeLa, and MCF-7). Mechanism-of-action studies demonstrated that 15u effectively inhibited tubulin polymerization, disrupted microtubule networks, arrested the cell cycle at the G2/M phase, and triggered apoptosis in cancer cells. In vivo, 15u significantly suppressed tumor growth in an Huh7 orthotopic mouse model without observable systemic toxicity. Taken together, these results identify 15u as a promising colchicine-site tubulin inhibitor with potent antitumor efficacy, supporting its further development as a potential anticancer agent.