Combretastatin A4 (CA4) is a novel vascular disrupting agent that has promising clinical efficacy because of its ability to inhibit microtubule assembly and subsequently disrupt tumor blood flow. In this study, we demonstrate that mitogen-activated protein kinases (MAPKs) are critically involved in the cytotoxicity of CA4. CA4 stimulates both extracellular signal-regulated kinases (ERK1/2) and p38 MAPK in the BEL-7402 hepatocellular carcinoma cell line in a time- and dose-dependent manner. This stimulation is a result of CA4-induced microtubule disassembly, which is a reversible process. Reversibility of microtubule disassembly is evidenced by the ability of disassembled microtubules to reassemble just a few hours after CA4 treatment. p38 MAPK, but not ERK1/2, contributes to this microtubule reassembly following CA4 exposure, and only inhibition of p38 MAPK, but not ERK1/2, synergistically enhances CA4-induced G(2)/M cell cycle arrest. Consistent with this, p38 MAPK inhibitors such as SB203580 and SB202190 also synergistically enhance the cytotoxicity of CA4 in cells where p38 MAPK is activated by CA4. This enhancement appears to be specific for CA4 because the cytotoxicity of other microtubule-targeted agents such as paclitaxel, vinorelbine and colchicine was not affected by p38 MAPK inhibitors. These data indicate that p38 MAPK is a potential anticancer target and that the combination of CA4 with p38 MAPK inhibitors may be a novel and promising strategy for cancer therapy.