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Summary Tubulin is one of the best validated anti-cancer targets, but most anti-tubulin agents have unfavorable therapeutic indexes. Here, we characterized the tubulin-binding activity, the mechanism of action, and the in vivo anti-leukemia efficacy of three 3,4,5-trimethoxy-N-acylhydrazones. We show that all compounds target the colchicine-binding site of tubulin and that none is a substrate of ABC transporters. The crystal structure of the tubulin-bound N-(1′-naphthyl)-3,4,5-trimethoxybenzohydrazide (12) revealed steric hindrance on the T7 loop movement of β-tubulin, thereby rendering tubulin assembly incompetent. Using dose escalation and short-term repeated dose studies, we further report that this compound class is well tolerated to >100 mg/kg in mice. We finally observed that intraperitoneally administered compound 12 significantly prolonged the overall survival of mice transplanted with both sensitive and multidrug-resistant acute lymphoblastic leukemia (ALL) cells. Taken together, this work describes promising colchicine-site-targeting tubulin inhibitors featuring favorable therapeutic effects against ALL and multidrug-resistant cells., Graphical Abstract, Highlights • 3,4,5-trimethoxy-N-acylhydrazones bind to the colchicine site of tubulin • 12 forms a single H-bond with αThr179 and causes steric hindrance of tubulin βT7 loop • 3,4,5-trimethoxy-N-acylhydrazones feature low toxicity • 12 shows therapeutic effect against multidrug-resistant acute lymphoblastic leukemia, Drugs; Biological Sciences; Molecular Biology; Structural Biology; Cancer