Abstract: Arabidopsis thaliana DnaJ homolog 3 (AtJ3), an Arabidopsis chaperone, interacts with SOS2-like protein kinase 5 (PKS5) to form the AtJ3-PKS5 complex and achieves its function through repressing activity of PKS5 in vivo. Moreover, the AtJ3-PKS5 complex positively regulates plasma membrane H⁺-ATPase activity and is involved in exogenous ABA response in Arabidopsis. In this study, salt- and ABA-treated Arabidopsis AtJ3 and PKS5 mutants were explored to elucidate the function and mechanism of the AtJ3-PKS5 complex in the simultaneous regulation of membrane H⁺-ATPase activity and ABA response. Results showed that the AtJ3-PKS5 complex not only led to changes in the cytosolic pH value via regulation of plasma membrane H⁺-ATPase activity, but also activated expression of the ABA-related responsive genes under the two treatments. Additionally, exogenous application of ABA induced trafficking of AtJ3 from the nucleus to the cytoplasm to enhance H⁺-ATPase activity, indicating that the AtJ3-PKS5 complex functions in ABA-meditated pH homeostasis. Taken together, our results suggest that the AtJ3-PKS5 complex might serve as a key regulator in a crosstalk metabolic pathway of H⁺-ATPase activity regulation and exogenous ABA response.