ETHNOPHARMACOLOGICAL RELEVANCY: Danggui Niantong Decoction (DGNTD) is a traditional Chinese medicine compound formula that has been demonstrated to possess efficacy in the treatment of rheumatoid arthritis (RA) and osteoarthritis (OA), as well as for dispelling moisture and relieving pain. As mentioned before, DGNTD is essential for synovial inflammation in RA. The primary features of the OA synovial membrane are low-grade inflammation, hyperplasia with enhanced fibroblast-like synoviocytes (FLS) proliferation, and fibrosis, which can cause pain and stiffness. However, it is still unknown how DGNTD functions in the OA synovium. AIM OF THE STUDY: Clarify the influence of DGNTD on OA synovium and investigate potential mechanisms of action. METHODS AND MATERIALS: The principal constituents of DGNTD were detected using liquid chromatography-mass spectrometry (LC-MS) analysis. To evaluate the effect of DGNTD on synovial inflammation and fibrosis, a transforming growth factor beta (TGF-β)-stimulated rat FLS cell model and a rat OA animal model based on anterior cruciate ligament transection (ACLT) and partial medial meniscectomy (MMx) were employed. RESULTS: Our results showed that 322 components were detected using LC-MS. In vivo, DGNTD therapy reduced pain, synovial inflammation, and fibrosis. The therapy significantly reduced levels of pain-related molecules, specifically calcitonin gene-related peptide (CGRP) and inducible nitric oxide synthase (iNOS), as well as fibrotic markers, including alpha smooth muscle actin (α-SMA) and type III collagen alpha-1 (Col3a1), in the synovium. A proteomics study demonstrated that DGNTD decreased the fibrotic protein Col3a1. DGNTD reduced the mRNA expression of pro-inflammatory and fibrotic markers (tumor necrosis factor alpha (TNF-α), interleukin-1 beta(IL-1β), interleukin-6(IL-6), α-SMA, Col3a1and TGF-β) in TGF-β-induced FLS. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and validation results revealed that DGNTD inhibits synovial fibrosis via the phosphatidylinositol 3-kinase (PI3K)/protein Kinase B (AKT) signaling pathway. CONCLUSIONS: DGNTD partially relieves pain, synovitis, and synovial fibrosis by regulating the PI3K/AKT pathway. These findings provide fresh information about the underlying mechanisms and successful therapy of OA, as well as a theoretical and experimental foundation for the clinical management of OA using DGNTD.