mHA11, a 2-amino-4-phenyl-4H-chromene-3-carboxylate analog, is a microtubule-targeting agent discovered by our group through the modification of the Bcl-2 inhibitor HA14-1. mHA11 exhibits cytotoxicities against tumor cells with nM IC50 values, whereas it has only a minimal effect on normal cells. We explored the plasma pharmacokinetics, tissue distribution, and excretion of mHA11 in rats using a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method. Next, we identified the metabolites of mHA11 and assessed the influence of cytochrome P450 (CYP) isozymes on mHA11 metabolism. We also examined the in vitro stability in rat plasma and rat liver microsomes (RLMs), the blood-to plasma (B/P) ratio, and the inhibitory effect on CYP isozyme activities. After oral administration at 5, 15, and 45 mg/kg, mHA11 was absorbed and eliminated rapidly. There was a linear correlation between the area under the concentration-time curve (AUC(0-infinity)) and the dose (R-2 = 0.983). The bioavailability of mHA11 was 4.1% at the oral dose of 15 mg/kg mHA11 was extensively distributed in various tissues and exhibited a high penetration into the brain. No significant parent drug was detected in urine or bile, and only 0.74% was recovered in feces, whereas two demethylated metabolites, M1 and M2, were found in the urine and feces, and further studies showed that CYP2C19 primarily contributed to metabolites formation. mHA11 was stable in rat plasma but degraded significantly in RLMs; its B/P ratio was 1.05 in rat blood. In addition, mHA11 dose-dependently inhibited the activities of rat CYP isozymes, including CYP1A2, CYP2C6, CYP2C11, CYP2D2, CYP2E1 and CYP3A2. The present study is the first report on the disposition of mHA11 in rats and provides important data for further research and development of this inhibitor.