Influenza viruses represent a serious threat to human health. Although our research group has previously demonstrated the antiviral and anti-inflammatory activities of eleutheroside B1, a detailed explanation of the mechanism by which it is effective against the influenza virus remains to be elucidated. In the present study, the transcriptomic responses of influenza A virus-infected lung epithelial cells (A549) treated with eleutheroside B1 were investigated using high-throughput RNA sequencing, and potential targets were identified using a molecular docking technique, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay, and DNA methylation analysis. The transcriptomic data revealed that there are 1,871 differentially expressed genes (DEGs) between the cells infected with the influenza virus strain variant PR8, and the cells infected with PR8 and treated with eleutheroside B1. Among the DEGs, RNA polymerase II subunit A (POLR2A; encoding the largest subunit of RNA polymerase II) and mannosidase class II member 1 (MAN2A1) were selected from the molecular docking analysis with eleutheroside B1. The docking score of Drosophila melanogaster MAN2A1 (3BVT) was 11.3029, whereas that of POLR2A was 9.0133. The RT-qPCR results demonstrated that the expression levels of host genes (MAN2A2, POLR2A) and viral genes (PA, PB1, PB2, HA) were downregulated following eleutheroside B1 treatment. Bisulfite-sequencing PCR was performed to investigate whether eleutheroside B1 was able to modify the DNA methylation of POLR2A, and the results suggested that the average proportion of methylated CpGs (-222-72 bp) increased significantly following treatment with eleutheroside B1. Taken together, these findings suggested that eleutheroside B1 may affect N-glycan biosynthesis, the chemokine signaling pathway, cytokine-cytokine receptor interaction and, in particular, may target the POLR2A to inhibit the production of influenza virus genes.