Background and Aims: Liver fibrosis, when dysregulated, contributes to irreversible loss of hepatic structure and function heralding end-stage liver diseases including cirrhosis and hepatocellular carcinoma. Hepatic stellate cells (HSCs) represent the common progenitor to myofibroblasts that produce extracellular matrix (ECM) proteins to mediate liver fibrosis. In the present study, we investigated the role of Pre-B-Cell leukemia transcription factor 1 (PBX1) in this process. Methods: Liver fibrosis was induced by CCl 4 injection or bile duct ligation (BDL). Cellular transcriptome was examined by RNA-seq and CUT&Tag-seq. Results: PBX1 was screened out of mining through liver fibrosis-related single-cell RNA-seq datasets as the most prominently up-regulated transcription factor during HSC activation. Ingenuity pathway analysis (IPA) coupled with reporter assay and ChIP assay demonstrated that Notch3 mediated elevation of PBX1 expression at the transcriptional level in HSCs. PBX1 knockdown attenuated HSC-myofibroblast transition in vitro and liver fibrosis in mice. Integrated transcriptomic analysis suggested that PBX1 contributed to HSC activation by activating a host of pro-fibrogenic molecules including interleukin 7 receptor (IL7R/CD127). Consistently, exposure to recombinant IL-7 promoted HSC activation whereas IL7R knockdown hampered HSC activation. Mechanistically, IL7R interacted with TGF-β receptors (TβRI/II) to trigger a pro-fibrogenic signaling cascade. Importantly, blockade of the IL-7-IL7R signaling with neutralizing antibodies markedly ameliorated liver fibrosis in mice. Finally, a positive correlation between PBX1, IL7R and HSC activation was identified in cirrhotic patients. Conclusions: Our data uncover a previously unappreciated role for PBX1 in HSC activation and provide proof-of-concept for targeting IL-7 signaling in the intervention of liver fibrosis.