Glabridin is a flavonoid compound characterized by diverse biological properties, such as anti-inflammatory, antioxidant, antitumor, and antibacterial activities. This study aimed to investigate the antiviral potential of Glabridin against influenza A virus (IAV), specifically focusing on the H1N1 strain. The qRT-PCR, Western blot analysis, and plaque assays were employed to evaluate the in vitro antiviral effects of Glabridin on H1N1. Furthermore, network pharmacology and molecular docking analyses were employed to predict the mechanisms underlying Glabridin antiviral action against IAV. The results revealed that Glabridin inhibited H1N1 virus replication in a concentration-dependent manner and exhibited significant antiviral activity (P<0.05). Pharm Mapper, Swiss Target Prediction database and Genecards database were further utilized to screen the intersection targets of Glabridin on IAV infection, and 161 overlapping targets were finally selected. R software version 4.3.1 was used to perform gene ontology (GO) enrichment and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses on these 161 overlapping targets. The results indicated that the overlapping targets were primarily implicated in processes such as the positive regulation of protein tyrosine kinase activity, the MAPK cascade, and the modulation of inflammatory responses. Moreover, these targets were enriched in various signaling pathways, including Kaposi's sarcoma-associated herpesvirus infection and the Rap1 signaling pathway. Venny software and String database were employed to construct the protein-protein interaction (PPI) network of the above-mentioned 161 overlapping targets, and a total of 10 potential core target proteins including SRC, EGFR, ESR1 were screened out. And the binding energies of Glabridin to all potential core targets were lower than -5.00 kcal/mol, indicating a high binding affinity. The results of this study indicated that Glabridin exhibits significant in vitro antiviral activity against the H1N1 virus, and its mechanism of action has the characteristics of multi-target, multi-pathway and multi-link regulation, which provides a reference for the elucidation of important functional proteins and the action mechanism of Glabridin against IAV.