<p>Herb pair of Acorus tatarinowii Schott (ATS) and Polygala tenuifolia Willd. (PTW) is a classic drug pair in the treatment of Alzheimer’s disease (AD), However, the mechanism by which the drug pair acts on AD is currently unknown. To address this, we constructed a PC12 cellular AD model using amyloid-beta peptide (Aβ) (25–35), follow by treating with different concentrations of ATS and PTW alone or their combination (1:1). The cell viability and Aβ-40, Aβ-42 and AQP4 expression were detected. In addition, RNA-sequencing combined with network pharmacology was performed to investigate the action mechanism of ATS and PTW, and the results were validated using in vitro experiments. The results showed that at drug-acting concentrations less than 100&#xa0;mg/L, both single-agent and combined treatments of ATS and PTW increased the protective effects on PC12 cell, and the herb pair was superior to single-agent. In addition, both single-agent and combined treatments of ATS and PTW (at concentration of 100&#xa0;mg/L) decreased Aβ-40, Aβ-42 and AQP4 expression compared with AD model. Further RNA-sequencing combined with network pharmacology analysis suggested that the underline action mechanism might be associated with Nos2-mediated calcium signaling pathway regulated. In vitro validation experiments showed that <i>Nos2</i> overexpression increase the levels of Aβ-40, Aβ-42, AQP4, p-Tau, CaM, and p-CaMKII, which were reversed by the combination treatment of ATS and PTW. In conclusion, this work indicates that ATS and PTW combination might alleviate an Aβ-induced cellular model through regulating <i>Nos2</i> - mediated calcium signaling pathway.</p> Graphical abstract <p></p>