2,4-Dichlorophenoxyacetic acid (2,4-D), the most widely used herbicide in the world, has been previously reported to induce lung damage. Here in this study we have investigated the molecular mechanism of 2,4-D induced lung toxicity in A549 and WI38 cell lines. Cell viability experiments indicate the IC50 values in A549 and WI38 cells for 72 h are 126 ± 2.25 μM and 115 ± 4.39 μM, respectively. Although not arresting a particular phase of the cell cycle of A549 cells, 2,4-D dose dependently increased the subG1 population, indicative of cell death, and the mode of cell death is apoptosis in both cell lines, as observed by annexin V/PI flow cytometric analysis and the expression status of pro and anti apoptotic proteins by western blot analysis. Dose dependent shrinkage of A549 cells indicates that 2,4-D can disrupt the microtubule network, and this is confirmed by immunofluorescence studies in vitro. In a cell free system, we found that 2,4-D depolymerises the microtubule network in vitro (IC50 = 212 ± 1.63 μM). 2,4-D can quench the intrinsic tryptophan fluorescence of tubulin in both a time and a dose dependent manner; the stoichiometry of 2,4-D-tubulin binding is 1:1 and the dissociation constant is 22.82 ± 1.29 μM. In silico studies indicate that 2,4-D binds to tubulin between the α and β subunits, very close to the colchicine binding site, and there is very little conformational change of the tubulin structure, as we also confirmed by circular dichroism studies. So, in brief, these results suggest that disruption of the cellular tubulin-microtubule network is one of the key mechanisms in the induction of lung cytotoxicity by 2,4-D. © 2014 The Royal Society of Chemistry.