Pulmonary fibrosis (PF) is a chronic interstitial lung disease with an average post-diagnosis survival of only 3-5 years. It is commonly characterized by dry cough and progressive dyspnea, ultimately leading to respiratory failure due to loss of gas exchange function. However, its exact pathogenic triggers and mechanisms remain unclear, and effective therapeutic drugs and approaches are still lacking. Therefore, further exploration of the pathogenesis of pulmonary fibrosis and the identification of new therapeutic targets are of great significance for its treatment and prognosis. Annexin A3 (ANXA3), a member of the calcium-dependent phospholipid-binding protein family, plays important roles in various pathophysiological processes such as membrane trafficking, inflammatory responses, and tumor progression. Nevertheless, its function and mechanism in pulmonary fibrosis have not yet been elucidated. In this study, using both in vivo animal models and in vitro cell experiments, we investigated the expression changes and functional mechanisms of ANXA3 in pulmonary fibrosis. In a bleomycin (BLM)-induced mouse model of pulmonary fibrosis, Western blot, quantitative real-time PCR, and immunofluorescence staining revealed that both the protein and mRNA levels of ANXA3 were significantly downregulated. To further clarify the role of ANXA3, a series of experiments were conducted using human embryonic lung fibroblasts (MRC-5). The results showed that knockdown of ANXA3 promoted the upregulation of fibrosis-related genes and significantly increased the expression of the extracellular matrix protein FN1. Meanwhile, EdU proliferation assays and wound healing assays demonstrated that ANXA3 knockdown markedly enhanced the proliferation and migration abilities of MRC-5 cells. In contrast, overexpression of ANXA3 significantly suppressed TGF-β1-induced fibrotic responses, including reduced expression of fibrotic markers, decreased extracellular matrix deposition, and weakened cell proliferation and migration. This study reveals that ANXA3 is downregulated during pulmonary fibrosis progression and plays a key inhibitory role in fibroblast activation and fibrogenesis, suggesting that ANXA3 may serve as an endogenous anti-fibrotic regulatory factor. These findings not only enrich the understanding of the ANXA protein family in the regulatory network of pulmonary fibrosis, but also provide experimental evidence for the future development of ANXA3-targeted therapeutic strategies for pulmonary fibrosis. This experiment was approved by the Ethics Committee of the School of Pharmacy, Harbin Medical University (approval number: IRB3035621).