Extracellular ATP (eATP) exists in the apoplast and plays multiple roles in growth, development, and stress responses. eATP has been revealed to suppresses growth rate and alters growth orientation of root and hypocotyl of Arabidopsis thalianaseedlings by affecting auxin transport in these organs. However, the mechanism of eATP-stimulated auxin distribution remains unclear. Annexins are involved in multiple aspects of plant cellular metabolism, while the role of annexins in response to apoplast signal remains unclear. Here, by using loss-of-function mutants, we investigated the role of several annexins in eATP-regulated root and hypocotyl growth. Since mutants of AtANN3 did not respond to eATP sensitively, the role of AtANN3 in eATP regulated auxin transport was intensively investigated. Firstly, the inhibitory effect of eATP on root or hypocotyl elongation was weakened or impaired in AtANN3 null mutants (atann3-1 and atann3-2). Meanwhile, single-, double- or triple-null mutant of AtANN1, AtANN2or AtANN4 responded to eATP in same manner and degree with Col-0. The distribution of DR5-GUS and DR5-GFP indicated that eATP-induced asymmetric distribution of auxin in root tip or hypocotyl cells, which appeared in wild type controls, were lacking in atann3-1seedlings. Further, eATP-induced asymmetric distribution of PIN2-GFP in root tip cells or PIN3-GFP in hypocotyl cells were reduced in atann3-1 seedlings. Based on these results, we suggest that AtANN3 may be involved in eATP-regulated seedling growth through regulating auxin transport in vegetative organs.