Abstract Background: Targeting tumor microenvironment (TME) may provide therapeutic activity and selectivity in treating cancers. Therefore, an improved understanding of the mechanism by which drug targeting TME would enable more informed and effective treatment measures. Glycyrrhiza uralensis Fisch (GUF, licorice), a widely used herb medicine, which has shown promising immunomodulatory activity and anti-tumor activity. However, the molecular mechanism of this biological activity has not been fully elaborated.Methods: Here, potential active compounds and specific targets of licorice that trigger the antitumor immunity were predicted with a systems pharmacology strategy. Flow cytometry technique was used to detect cell cycle profile and CD8+ T cell infiltration of licorice treatment. And anti-tumor activity of licorice was evaluated in C57BL/6 mice.Results: We reported the G0/G1 growth phase cycle arrest of tumor cells induced by licorice that is related with the down-regulation of CDK4-Cyclin D1 complex, which subsequently led to increased protein abundance of PD-L1. Further, in vivo studies demonstrated that mitigation the outgrowth of NSCLC tumor induced by licorice was reliant on increased antigen presentation and improved CD8+ T cell infiltration.Conclusions: Briefly, our findings improved understanding of the anti-tumor effects of licorice with the systems pharmacology strategy, thereby promoting the development of natural products in the prevention or treatment of cancers.Trial registration: Not applicable