Background <p>The therapeutic efficacy of programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) inhibitors in lung cancer management remains suboptimal. Sporoderm-removed Ganoderma Lucidum Spore Powder (RGLS), an immunostimulant, has been shown to amplify the anti-tumor effects of anti-PD-L1 antibodies (αPD-L1). However, the detailed molecular underpinnings of this enhancement are not yet fully elucidated. This study aimed to elucidate the antitumor mechanism and immunomodulatory effect of RGLS in additive effect relationship with αPD-L1 in lung cancer.</p> Methods <p>Employing network pharmacology approaches, this research analyzed the multifaceted target pathways of RGLS in lung cancer. High-Performance Liquid Chromatography (HPLC) facilitated the identification of RGLS constituents. In the established Lewis lung cancer tumor-bearing mouse model, the effects of RGLS, αPD-L1, and their combination were investigated, focusing on tumor growth, T cell responses, and the dynamics of myeloid-derived suppressor cells (MDSCs) within tumor microenvironments.</p> Results <p>Our network analysis revealed 26 bioactive components of RGLS and identified 227 potential lung cancer targets, among which PD-L1 had a significant response effect with 20 core targets. HPLC identified 14 triterpenoid components. Notably, the combination of RGLS and αPD-L1 significantly inhibited tumor growth, optimized the CD4⁺/CD8⁺ T cell ratio in tumor tissue, increased IL-2 and IFN-γ levels, enhanced cytotoxic T lymphocyte (CTL) activity, and inhibited MDSCs recruitment.</p> Conclusion <p>The additive effect application of RGLS and αPD-L1 in lung cancer treatment significantly improves immune responsiveness and reduces tumor-associated MDSCs, surpassing the efficacy of sole αPD-L1 application. This study underscores RGLS’s burgeoning potential in bolstering lung cancer immunotherapy, paving the way for its clinical applications.</p>