Microtubule-Targeting agents (MTA) are indispensable for cancer therapeutics. We here report thymoquinone (TQ) as a new MTA that already has been appreciated for its anticancer effects. TQ induced G2/M cell cycle arrest in human non-small lung epithelial cells (A549) and majority of arrested cells were in mitosis. TQ depolymerized the microtubule (MT) network and disrupted mitotic spindle organization of A549 cells. MT depolymerization by TQ was followed by apoptosis and subsequent loss in cell viability (IC 50 value of ∼10 μM). Interestingly, TQ didn't affect the MT network of normal HUVEC cells at and below the IC50 concentration for A549 cells. TQ also inhibited tubulin polymerization in cell-free system with an IC50 of 27 μM and bound to tubulin heterodimers at a single site with a dissociation constant of 1.19 μM at 25 C. Binding of TQ to tubulin quenched the tryptophan fluorescence of protein in a time-dependent manner. The TQ-tubulin binding kinetics was biphasic in nature and equilibrated in 30 min. TQ competed with colchicine for tubulin binding with a Ki of 1.9 μM as determined by modified Dixon plot analysis, this suggests TQ may bind tubulin at or near the colchicine binding site and in silico modeling study supported that. Our results establish a novel antimitotic mechanism of TQ by its direct binding to tubulin-MT network in A549 cells. © 2013 Elsevier Masson SAS. All rights reserved.