SummaryUnlike conventional lysine (K) 48–linked polyubiquitination, K63‐linked polyubiquitination plays signaling roles in yeast and animals. Thus far, UBC13 is the only known ubiquitin‐conjugating enzyme (E2) specialized in K63‐linked polyubiquitination. Previous identification of Arabidopsis genes encoding UBC13 as well as its interacting partner UEV1 indicates that the UBC13‐mediated ubiquitination pathway is conserved in plants; however, little is known about functions and signaling mediated through K63‐linked polyubiquitination in plants. To address the functions of UBC13‐mediated ubiquitination in plants, we created Arabidopsis ubc13 null mutant lines in which the two UBC13 genes were disrupted. The double mutant displayed altered root development, including shorter primary root, fewer lateral roots and only a few short root hairs in comparison with the wild type and single mutant plants, indicating that UBC13 activity is critical for all major aspects of root development. The double mutant plants were insensitive to auxin treatments, suggesting that the strong root phenotypes do not simply result from a reduced level of auxin. Instead, the ubc13 mutant had a reduced auxin response, as indicated by the expression of an auxin‐responsive DR5 promoter‐GFP. Furthermore, both the enzymatic activity and protein level of an AXR3/IAA17‐GUS reporter were greatly increased in the ubc13 mutant, whereas the induction of many auxin‐responsive genes was suppressed. Collectively, these results suggest that Aux/IAA proteins accumulate in the ubc13 mutant, resulting in a reduced auxin response and defective root development. Hence, this study provides possible mechanistic links between UBC13‐mediated protein ubiquitination, root development and auxin signaling.