Osteoarthritis (OA) is a common chronic age-related joint disease characterized primarily by inflammation of synovial membrane and degeneration of articular cartilage. Accumulating evidence has demonstrated that Danggui-Shaoyao-San (DSS) exerts significant anti-inflammatory effects, suggesting that it may play an important role in the treatment of knee osteoarthritis (KOA).In the present study, DSS was prepared and analyzed by high-performance liquid chromatography (HPLC). Bioinformatics analyses were carried out to uncover the functions and possible molecular mechanisms by which DSS against KOA. Furthermore, the protective effects of DSS on lipopolysaccharide (LPS)-induced rat chondrocytes and cartilage degeneration in a rat OA model were investigated in vivo and in vitro.In total, 114 targets of DSS were identified, of which 60 candidate targets were related to KOA. The target enrichment analysis suggested that the NF-κB signaling pathway may be an effective mechanism of DSS. In vitro, we found that DSS significantly inhibited LPS-induced upregulation of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), matrix metalloproteinase-3 (MMP3), and matrix metalloproteinase-13 (MMP13). Meanwhile, the degradation of collagen II was also reversed by DSS. Mechanistically, DSS dramatically suppressed LPS-induced activation of the nuclear factor kappa B (NF-κB) signaling pathway. In vivo, DSS treatment prevented cartilage degeneration in a rat OA model.DSS could ameliorate the progression of OA through suppressing the NF-κB signaling pathway. Our findings indicate that DSS may be a promising therapeutic approach for the treatment of KOA.