SummaryThe balance between carbon (C) and nitrogen (N) availability is an important determinant for various phases of plant growth; however, the detailed mechanisms regulating the C/N response are not well understood. We previously described two related ubiquitin ligases, ATL31 and ATL6, that function in the C/N response in Arabidopsis thaliana. Here, we used FLAG tag affinity purification and MS analysis to identify proteins targeted by ATL31, and thus likely to be involved in regulating the phase transition checkpoint based on C/N status. This analysis revealed that 14‐3‐3 proteins were associated with ATL31, and one of these, 14‐3‐3χ, was selected for detailed characterization. The interaction between ATL31 and 14‐3‐3χ was confirmed by yeast two‐hybrid and co‐immunoprecipitation analyses. In vitro assays showed that ubiquitination of 14‐3‐3χ is catalyzed by ATL31. Degradation of 14‐3‐3χin vivo was shown to be correlated with ATL31 activity, and to occur in a proteasome‐dependent manner. Furthermore, 14‐3‐3 protein accumulation was induced by a shift to high‐C/N stress conditions in Arabidopsis seedlings, and this regulated response required both ATL31 and ATL6. It was also shown that over‐expression of 14‐3‐3χ leads to hypersensitivity of Arabidopsis seedlings to C/N stress conditions. These results indicate that ATL31 targets and ubiquitinates 14‐3‐3 proteins for degradation via the ubiquitin–proteasome system during the response to cellular C/N status.