Microtubule targeting agents (MTAs) are extensively used in cancer treatment and many have achieved substantial clinical success. In recent years, targeting microtubules to inhibit cell division has become a widespread pharmaceutical approach for treatment of various cancer types. Nevertheless, the development of multidrug resistance (MDR) in cancer remains a major obstacle for successful application of these agents. Herein, we provided the evidence that CKT0353, α-branched α,β-unsaturated ketone, possesses the capacity to successfully evade the MDR phenotype as an MTA. CKT0353 induced G2/M phase arrest, delayed cell division via spindle assembly checkpoint activation, disrupted the mitotic spindle formation and depolymerized microtubules in human breast, cervix, and colorectal carcinoma cells. Molecular docking analysis revealed that CKT0353 binds at the nocodazole binding domain of β-tubulin. Furthermore, CKT0353 triggered apoptosis via caspase-dependent mechanism. In addition, P-glycoprotein overexpressing colorectal carcinoma cells showed higher sensitivity to this agent when compared to their sensitive counterpart, demonstrating the ability of CKT0353 to overcome this classic MDR mechanism involved in resistance to various MTAs. Taken together, these findings suggest that CKT0353 is an excellent candidate for further optimization as a therapeutic agent against tumors with MDR phenotype.