Pulmonary fibrosis is a manifestation of the progression of interstitial pulmonary disease. Icariin (ICA) has been found to exhibit protective effects on multiple chronic diseases like diabetes, liver, heart, and renal fibrosis. Here, a systemic pharmacological study was designed to investigate whether ICA treatment alleviates bleomycin (BLM)-induced pulmonary fibrosis. The rat pulmonary fibrosis model was constructed by non-invasive endotracheal intubation instillation of BLM to observe the intervention effects of ICA on pulmonary fibrosis in the whole process of inflammation and fibrosis. ICA reduced the collagen deposition and inflammation induced by BLM in rat. The comparative RNA-sequencing was conducted to analyze the lung gene expression profiles in rat. KEGG analysis indicated that most of the genes were enriched in Hippo pathway, NF-κB pathway, and B-cell receptor signaling pathway, etc. Immunohistochemistry staining showed that the expression of YAP was significantly elevated in the model group and decreased in the ICA treatment group. Taken together, the anti-fibrotic effect of ICA appears to be mediated by its inhibitory of YAP, which is the core transcriptional regulator of Hippo pathway.