The synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) functions as an agronomic weed control herbicide. High concentrations of 2,4-D induce plant growth defects, particularly leaf epinasty and stem curvature. Although the 2,4-D-triggered ROS production, little is known about its signalling. In this study, by using a null mutant in peroxisomal acyl CoA oxidase 1 (acx1-2), we identified ACX1 as one of the main sources of ROS production and, in part, also causing the epinastic phenotype following 2,4-D application. Transcriptomic analyses of WT plants after treatment with 2,4-D revealed a ROS-related peroxisomal footprint in early plant responses, while other organelles, such as mitochondria and chloroplasts, are involved in later responses. Interestingly, a group of 2,4-D-responsive ACX1-dependent transcripts previously associated with epinasty is related to auxin biosynthesis, metabolism and signalling. We found that the auxin receptor AUXIN SIGNALING F-BOX 3 (AFB3), a component of SCF (ASK-cullin-F-box) E3 ubiquitin ligase complexes, which mediates AUX/IAA degradation by the 26S proteasome, acts downstream of ACX1 and is involved in the epinastic phenotype induced by 2,4-D. We also found that protein degradation associated with ubiquitin E3-RING and E3-SCF-FBOX in ACX1-dependent signalling in plant responses to 2,4-D is significantly regulated over longer treatment periods. This article is protected by copyright. All rights reserved.