BackgroundImmune checkpoint inhibitors (ICIs) have altered the clinical management of non-small cell lung cancer (NSCLC). However, the low response rate, severe immune-related adverse events (irAEs), and hyperprogressive disease following ICIs monotherapy require attention. Combination therapy may overcome these limitations and traditional Chinese medicine with immunomodulatory effects provides a promising approach. Shenmai injection (SMI) is a clinically effective adjuvant treatment for cancer with chemotherapy and radiotherapy. Therefore, the combined effects and mechanisms of SMI and programmed death-1 (PD-1) inhibitor against NSCLC was focused on this study.MethodsA Lewis lung carcinoma mouse model and a lung squamous cell carcinoma humanized mouse model were used to investigate the combined efficacy and safety of SMI and PD-1 inhibitor. The synergistic mechanisms of the combination therapy against NSCLC were explored using single-cell RNA sequencing. Validation experiments were performed using immunofluorescence analysis, in vitro experiment, and bulk transcriptomic datasets.ResultsIn both models, combination therapy alleviated tumor growth and prolonged survival without increasing irAEs. TheGZMAhighandXCL1highnatural killer (NK) cell subclusters with cytotoxic and chemokine signatures increased in the combination therapy, while malignant cells from combination therapy were mainly in the apoptotic state, suggesting that mediating tumor cell apoptosis through NK cells is the main synergistic mechanisms of combination therapy. In vitro experiment confirmed that combination therapy increased secretion of Granzyme A by NK cells. Moreover, we discovered that PD-1 inhibitor and SMI combination blocked inhibitory receptors on NK and T cells and restores their antitumoral activity in NSCLC better than PD-1 inhibitor monotherapy, and immune and stromal cells exhibited a decrease of angiogenic features and attenuated cancer metabolism reprogramming in microenvironment of combination therapy.ConclusionsThis study demonstrated that SMI reprograms tumor immune microenvironment mainly by inducing NK cells infiltration and synergizes with PD-1 inhibitor against NSCLC, suggested that targeting NK cells may be an important strategy for combining with ICIs.Video AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s12964-023-01184-3.