Objective To investigate the regulation of β-adrenoceptor on sodium-calcium exchange current (INCX) and its possible signal transduction pathway. Methods The NCX1.1 plasmid was transfected into human embryonic kidney-293 (HEK-293) cell by immunomagnetic-bead-based positive expression system. Transfected HEK-293 cells totaling 103 with positive transfection were selected and randomly divided into blank control group (n=20), isoproterenol (ISO) group (n=20), pertussis toxin (PTX) plus ISO group (n=20), forskolin plus ISO group (n=22), and H89 [protein kinase A (PKA) inhibitor] plus ISO group (n=21). INCX changes in each group were recorded with whole-cell patch clamp technique. SPSS statistics 21.0 was used for statistical analysis. According to data type, t-test was used for comparison between two groups, and ANOVA was used for comparison between multiple groups. Results Against control group, ISO increased the inward INCX density from (-6.07±1.53) pA/pF to (-7.89±1.61) pA/pF (n=20, P<0.05), with an average increase of about 30%. However, the effect of ISO on INCX current was significantly changed after pre-administration of key molecular agonists or inhibitors of Gi-cAMP-PKA pathway. PTX and forskolin, significantly enhanced the effect of ISO, had a more significant effect on INCX density increase, the current density increased to (-10.02±1.99) pA/pF and (-10.78±1.77) pA/pF, respectively. Compared with ISO alone, there was a significant difference (n=20, P<0.01), suggesting that both the inhibition of Gi protein and activation of cAMP can enhance the effect of ISO. However, the effect of pre-treatment with PKA inhibitor H89 showed no enhancement on INCX , and the current density was about (-6.22±1.70) pA/pF, which was not significantly different from that of the control group (n=20,P>0.05). It suggested that PKA inhibition can basically block the effect of ISO on INCX. Conclusion β-adrenoceptor activation may enhance the NCX1.1 inward current, probably through stimulating G protein-cAMP-PKA signaling pathway.