Objective: To investigate the effects of grape seed extract (GSE) on NF-κB expression and inflammatory injury in synoviocytes of mice with rheumatoid arthritis (RA), so as to provide experimental evidence for the natural drug therapy of RA. Methods: RA mouse model was established by collagen induction. The mice were randomly divided into a normal control group, a model group, and low-/medium-/high-dose GSE groups, and received intragastric intervention for 48 hours. Synoviocytes were isolated and cultured in vitro. The mRNA and protein expressions of NF-κB/p65 were detected by qRT-PCR and Western blot, respectively. The secretion levels of TNF-α, IL-6, and IL-1β were detected by ELISA, and the pathological changes of synovial tissue were observed by HE staining. Results: Compared with the model group, the expression of NF-κB/p65 in each GSE dose group decreased in a dose-dependent manner (P < 0.05 or P < 0.01). The expression and secretion of downstream inflammatory factors were significantly down-regulated, and the pathological injury of synovial tissue was significantly improved, with the most significant effect in the high-dose group. Conclusion: GSE can inhibit the NF-κB signaling pathway, down-regulate the release of inflammatory factors, and alleviate synovial inflammation and pathological injury in RA mice. It is expected to be a potential natural drug for the treatment of RA.