Targeting the colchicine binding site on tubulin is a promising strategy to develop cancer therapeutics. Herein, we describe our systematic structure-activity relationship studies of benzamide derivatives that lead to an identification of a potent and orally active tubulin inhibitor <b>48</b>, which occupied all three zones of the colchicine binding site in the X-ray co-crystal structure, inhibited tubulin polymerization, promoted mitotic blockade and apoptosis, and exhibited significant antiproliferative activities against various cancer cell lines. Compound <b>48</b> demonstrated favorable pharmacokinetic profiles, robust <i>in vivo</i> antitumor efficacies, and acceptable safety profiles. Furthermore, <b>48</b> overcame drug resistance in the paclitaxel-resistant A549 xenograft model. Collectively, <b>48</b> has been advanced into further preclinical evaluation for the development of next-generation microtubule-targeting drugs.