Lycorine (LYC), the most abundant monomeric compound from Lycoris radiata bulbs, possesses a range of biological activities, including antipulmonary fibrosis and cardioprotective effects. However, its role in regulating pathological cardiac remodeling, particularly in cardiac fibrosis, remains unclear. In this study, we used transforming growth factor-β1 (TGF-β1) to stimulate NIH/3T3 cells and primary rat cardiac fibroblasts, which were in vitro models of pathological fibroblast activation, and established a mouse model of cardiac fibrosis via intraperitoneal injection of doxorubicin (DOX). The effects of LYC on TGF-β1-induced fibroblast activation and migration were assessed using Western blotting, quantitative PCR, scratch assays, and EdU staining. Cardiac damage and fibrosis were evaluated in vivo by echocardiography, enzyme-linked immunosorbent assay (ELISA), and Masson's trichrome staining. Through transcriptomic sequencing and bioinformatic analyses, proline-rich tyrosine kinase 2 (PYK2) was identified as a potential target of LYC. This finding was further validated using siPYK2 and the PYK2 inhibitor PF4618433 in TGF-β1-induced NIH/3T3 cells. Our results demonstrated that LYC effectively suppressed fibroblast activation, proliferation, and migration. In addition, LYC significantly ameliorated cardiac dysfunction and reduced collagen deposition in myocardial tissues, thereby attenuating the progression of cardiac fibrosis. Mechanistically, these effects were attributed to the regulation of PYK2 expression and activity by LYC. This study provides a scientific basis for understanding the mechanism of LYC in improving cardiac fibrosis and highlights PYK2 as a potential therapeutic target for the treatment of cardiac fibrosis.