Background and Purpose: The incidence of liver fibrosis has remained high worldwide, posing a serious threat to human health. Carthamus tinctorius L is a traditional medicine for treating liver disease, and flavonoids, as the main active ingredients, have a wide range of pharmacological activities. This study investigated the pharmacodynamic effects and mechanism of action of total flavonoids from Carthamus tinctorius L (TFCTL) on hepatic fibrosis mice and TGF-β1 induced activated hepatic stellate cells (HSC). Methods: LC-MS/MS technique was used to identify the chemical constituents of TFCTL. We established an animal model of liver fibrosis and simultaneously induced the activation of HSC-T6 cells in vitro and CCK-8, Western blot, flow cytometry, RT-qPCR, immunofluorescence technology were applied to investigate the anti-hepatic fibrosis effect and mechanism of TFCTL. Results: TFCTL can promote YAP phosphorylation and degradation by inhibiting the activation and proliferation of HSC-T6 cells in vitro, increasing the expression of MST1 and LATS1, and then inhibiting the expression of downstream target genes in the Hippo signaling pathway. TFCTL can significantly improve the pathological conditions of liver fibrosis mice and the mechanism of action is mainly related to the Hippo/YAP pathway. Implications: TFCTL has significant anti-fibrotic effects which may be recognized as a prospective drug candidate for the therapy of liver fibrosis.