Abstract: Objective To investigate the regulation and possible mechanism of H₂O₂ on the inward current of Na⁺/Ca²⁺-exchanger（NCX）. Methods The NCX1.1 plasmid was transfected into human embryonic kidney cells（HEK293 cell） by green fluorescent protein labeling expression system. The whole cell patch clamp technique was used to record NCX current, and the concentration-dependent effect of H₂O₂ on the current and Ⅰ-Ⅴ curve of NCX1.1 were recorded. A total of 120 positive HEK293 cells were selected and divided into control group, H₂O₂ treatment group, pertussis toxin（Gi protein inhibitor） and H₂O₂ treatment group. inhibitor H89（protein kinase A） and H₂O₂ treatment. Analysis of I_NCX before and after treatment. Western-blot assay was used to detect the changes of NCX1.1 protein level in H₂O₂ and HEK293 cell group, PTX+H₂O₂ and HEK293 cell group, H89+ H₂O₂ and HEK293 cell group after 24 h culture. Results H₂O₂increased the inward current of I_NCX from（-6.22±0.53） to（-12.92±1.12） pA/pF(n=60, t=2.54, P＜0.01, and the inward current of NCX1.1 was increased in a concentration-dependent manner by extracellular perfusion with different concentrations of H₂O₂. The results of inverted ramp stimulation mode showed that the effect of H₂O₂ on the inward current of NCX1.1 was voltage dependent. The selective inhibitor of Gi protein PTX was added, and PTX was found to reverse the increase effect of H₂O₂H₂O₂:（-12.92±1.12） vs H₂O₂+PTX:（-6.76±0.60）pA/pF, n=60, t=3.61, P＜0.001. The PKA inhibitor H89 also reversed the increase effect of H₂O₂H₂O₂:（-12.92±1.12） vs H₂O₂+H89:（-7.22±1.60）pA/pF, n=60, t=4.17, P＜0.005. In addition, the expression of NCX1.1 protein in HEK293 cells was increased after 24h culture with H₂O₂. However, PTX and H89 can reverse the increasing effect of H₂O₂ on the expression of NCX1.1 protein. Conclusion H₂O₂ stimulation can increase the inward current of NCX1.1, and this process is mediated by the Gi-cAMP-PKA signaling pathway.