14-3-3 pproteins play essential roles in diverse cellular processes through the direct binding to target proteins. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcription factor that is involved in gibberellin (GA) feedback regulation. The 14-3-3 proteins bind to RSG depending on the RSG phosphorylation of Ser-114 and negatively regulate RSG by sequestering it in the cytoplasm in response to GAs. The Ca 2+ -dependent protein kinase NtCDPK1 was identified as an RSG kinase that promotes 14-3-3 binding of RSG by phosphorylation of RSG. 14-3-3 weakly binds to NtCDPK1 by a new mode. The autophosphorylation of NtCDPK1 was necessary for the formation of the binding between NtCDPK1 and 14-3-3 but not for its maintenance. In this study, we showed that 14-3-3 binding to NtCDPK1 does not require the autophosphorylation when RSG was bound to NtCDPK1. These data suggest that 14-3-3 binds to an unphosphorylated motif in NtCDPK1 exposed by a conformational change in NtCDPK1 but not to a phosphate group generated by autophosphorylation of NtCDPK1.