Aluminium (Al) is phytotoxic when solubilized into Al3+ in acidic soils. One of the earliest and distinct symptoms of Al3+ toxicity is inhibition of root elongation. To decipher the mechanism by which Al3+ inhibits root elongation, the role of ethylene and auxin in Al3+-induced inhibition of root elongation in Arabidopsis thaliana was investigated using the wild type and mutants defective in ethylene signalling (etr1-3 and ein2-1) and auxin polar transport (aux1-7 and pin2). Exposure of wild-type Arabidopsis to AlCl3 led to a marked inhibition of root elongation, and elicited a rapid ethylene evolution and enhanced activity of the ethylene reporter EBS:GUS in root apices. Root elongation in etr1-3 and ein2-1 mutants was less inhibited by Al3+ than that in wild-type plants. Ethylene synthesis inhibitors, Co2+ and aminoethoxyvinylglycine (AVG), and an antagonist of ethylene perception (Ag+) abolished the Al3+-induced inhibition of root elongation. There was less inhibition of root elongation by Al3+ in aux1-7 and pin2 mutants than in the wild type. The auxin polar transport inhibitor, naphthylphthalamic acid (NPA), substantially alleviated the Al3+-induced inhibition of root elongation. The Al3+ and ethylene synthesis precursor aminocyclopropane carboxylic acid (ACC) increased auxin reporter DR5:GUS activity in roots. The Al3+-induced increase in DR5:GUS activity was reduced by AVG, while the Al3+-induced increase in EBS:GUS activity was not altered by NPA. Al3+ and ACC increased transcripts of AUX1 and PIN2, and this effect was no longer observed in the presence of AVG and Co2+. These findings indicate that Al3+-induced ethylene production is likely to act as a signal to alter auxin distribution in roots by disrupting AUX1- and PIN2-mediated auxin polar transport, leading to arrest of root elongation.