The boronic acid group plays an important role in drug discovery. Following our discovery of a boronic acid analog of combretastatin A-4 (CA-4), a series of analogs featuring a boronic acid group on the C phenyl ring of CA-4 was synthesized and evaluated for cytotoxicity, as well as for their ability to inhibit tubulin polymerization, inhibit the binding of [³H]colchicine to tubulin and cause depolymerization of cellular microtubules. Modifications on the C ring of CA-4, either eliminating the methoxy group or replacing the C phenyl ring with a pyridine ring, resulted in a reduced potency for inhibiting tubulin polymerization, colchicine binding and cytotoxic activities as compared to CA-4. Replacing the phenol group with a boronic acid group on the C ring of phenstatin led to a slight increase in cytotoxic potency but a decreased potency for inhibition of tubulin assembly and colchicine binding. Moreover, there was a significant decrease in activity by replacing the C phenyl ring with a pyridine ring. Our results indicate the critical importance of the methoxy group on the C ring as well as the importance of the C phenyl ring compared to a pyridine ring, despite the latter providing a nitrogen atom as a hydrogen bond donor/acceptor, which was predicted by molecular modeling to enhance interaction with the target. The decreased activities of our modified CA-4 boronic analogs may be attributed to weakened hydrogen bonding in our docking model based on the crystal structure of colchicine bound to αβ-tubulin. Notably, even though their effectiveness in inhibiting tubulin polymerization and colchicine binding and causing microtubule depolymerization in cells, the majority of these boronic acid analogs exhibited substantial cytotoxicity. This suggests that they may have additional cellular targets that contribute to their cytotoxicity, and this warrants further evaluation of these unique boronic acid compounds as potential anticancer agents.