Background <p>Rheumatoid arthritis (RA) is an autoimmune disorder characterized by synovial inflammation and bone erosion, yet key immune-driven molecular regulators remain incompletely defined.</p> Objective <p>This study aimed to identify differentially expressed genes (DEGs) in RA, explore their potential association with disease pathogenesis, and assess the potential of lymphocyte-specific protein tyrosine kinase (LCK) as a therapeutic target for RA.</p> Methods <p>Bioinformatic analysis was conducted using three microarray datasets from the Gene Expression Omnibus (GEO) database. Differentially expressed genes were identified and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. A protein–protein interaction (PPI) network was constructed to identify hub genes, with further validation using clinical samples, an animal model, and in vitro experiments. The effect of LCK inhibition on RA progression was explored using a collagen-induced arthritis (CIA) rat model.</p> Results <p>A total of 115 DEGs were identified across three GEO datasets, with significant enrichment in immune-related pathways. PPI analysis highlighted LCK, GZMA, GZMB, CD2, LAG3, and CD247 as hub genes, with LCK markedly upregulated in RA synovium. LCK is a Src-family tyrosine kinase that phosphorylates the TCR/CD3 complex to initiate T-cell receptor signaling and drive CD4⁺ T-cell activation. Inhibition of LCK in CIA rats led to reduced CD4⁺ Tcell activation, osteoclast activity, and alleviated bone destruction. In vitro, LCK inhibition reduced CD4⁺ T-cell activation, suppressed osteoclast differentiation and inflammatory cytokine secretion.</p> Conclusion <p>LCK may play an important role in RA by regulating with CD4⁺ T-cell activation and promoting osteoclast differentiation. Targeting LCK may represent a promising strategy to reduce inflammation and bone destruction in RA, suggesting its potential as a therapeutic target.</p>