RSG was involved in plant gibberellin feedback regulation by inducing the expression of key genes. The tobacco calcium-dependent protein kinase, CDPK1 was reported to interact with RSG and manipulate its intracellular localization by phosphorylating Ser-114 of RSG. Here, we identified tobacco mitogen-activated protein kinase 3 (NtMPK3) as an RSG interacted protein kinase. Mutation of predicted MAPK-associated phosphorylation site of RSG (Thr-30, Ser-74 and Thr-135) significantly altered the intracellular localization of NtMPK3-RSG interaction complex. Nuclear transport of RSG and its amino acids mutants (T30A and S74A) were observed after treated with plant defense elicitor peptide flg22 in 5 min, while the two mutated RSG swiftly relocalized in tobacco cytoplasm in 30 min. Moreover, triple points mutation of RSG (T30A/S74A/T135A) mimics constant unphosphorylated status, and predominantly localized in tobacco cytoplasm. RSG (T30A/S74A/T135A) showed no relocalization effect under the treatments of either flg22, B. cereus AR156 or GA3, and was impaired in its role as TFs. Our results suggest that MAPK associated phosphorylation sites of RSG regulate its localization in tobacco and constant unphosphorylation of RSG in Thr-30, Ser-74 and Thr-135 keeps RSG predominantly localized in cytoplasm.