ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniae Radix Alba (the root of Paeonia lactiflora Pall.) is a well-known Chinese herb medicine used for alleviating depression and anxiety. Paeoniflorin (PF), an active ingredient of Paeoniae Radix Alba, is usually used in emotion and inflammation-related diseases. In recent years, some studies showed that PF may also possess anti-tumor potential. AIM OF THE STUDY: This study aimed to explore the effects of PF on chronic restraint stress (CRS)-induced hepatocellular carcinoma (HCC) progression and elucidate the potential molecular mechanisms. MATERIALS AND METHODS: ICR male mice bearing H22-Luc orthotopic transplant tumors were subjected to CRS and administrated with PF. To identify the direct target of PF, network pharmacology, RNA sequencing, and molecular docking analyses were employed. CCK8, Western blotting and qRT-PCR assays were performed to explore the molecular mechanisms of PF. RESULTS: PF mitigated CRS-induced depression-like behaviors in tumor-bearing mice and suppressed the growth of orthotopically transplanted tumors. PF also decreased the number of c-fos positive neurons in the paraventricular nucleus of hypothalamus in CRS-exposed mice and lowered the serum norepinephrine (NE) level. NE treatment promoted the proliferation and αSMA production of hepatic stellate cells (HSCs), but did not alter the viability and migration of HCC cells. Furthermore, the conditional medium (CM) from NE-treated HSCs enhanced the proliferation and migration of HCC cells. PF not only inhibited NE-induced activation of HSCs, also reduced HSCs-CM induced viability and migration of HCC cells. Network pharmacology and RNA sequencing showed SRC was a potential target of PF in HSCs, which was further validated by molecular docking and cellular thermal shift assay. NE treatment upregulated the phosphorylation of SRC, AKT and ERK1/2 in HSCs, which was inhibited by PF. CONCLUSION: The findings of this study support that PF could ameliorate CRS-induced HCC progression by inhibiting CRS-induced HSCs activation through SRC/AKT/ERK signaling pathway. Our work may provide a new prospect for PF in the treatment of HCC with comorbid psychological stress.