We recently reported the crystal structure of tubulin in complex with a colchicine binding site inhibitor (CBSI), ABI-231, having 2-aryl-4-benzoyl-imidazole (ABI). Based on this and additional crystal structures, here we report the structure-activity relationship study of a novel series of pyridine analogues of ABI-231, with compound <b>4v</b> being the most potent one (average IC<sub>50</sub> ∼ 1.8 nM) against a panel of cancer cell lines. We determined the crystal structures of another potent CBSI ABI-274 and <b>4v</b> in complex with tubulin and confirmed their direct binding at the colchicine site. <b>4v</b> inhibited tubulin polymerization, strongly suppressed A375 melanoma tumor growth, induced tumor necrosis, disrupted tumor angiogenesis, and led to tumor cell apoptosis in vivo. Collectively, these studies suggest that <b>4v</b> represents a promising new generation of tubulin inhibitors.