Inhibition of p38 MAPK suppresses the expression of pro-inflammatory cytokines such as TNF and IL-1 in macrophages and fibroblast-like synoviocytes (FLS). However, there have been no genome-wide studies on the gene targets of p38 MAPK signaling in the synoviocytes. Microarray technology was applied in order to generate a comprehensive analysis of all genes regulated by the p38 MAPK signaling pathway in FLS. Gene expression levels were measured using Agilent oligonucleotide microarrays. Four independent sets of mRNA modulated by TNFand vehicle were used to measure the change of gene expression due to TNF and three experiments were done to ascertain the effect of SB203580, a p38 MAPK inhibitor, on TNF-induced genes. Microarray data were validated by RT-qPCR. 141 significantly expressed genes were more than two-fold upregulated by TNF. 30% of these genes were downregulated by the p38 inhibitor, SB203580, whereas 67% of these genes were not significantly changed by the inhibitor. The SB203580-inhibited genes include pro-inflammatory cytokines such as interleukins and chemokines, proteases including matrix metallopeptidases, metabolism-related genes such as cyclooxygenases and phosphodiesterase, genes involved in signal transduction, and genes encoding for transcription factors, receptors and transporters. Approximately one third of the TNF induced genes in FLS are regulated by the p38 MAPK signaling pathway, showing that p38 MAP kinase is a possible target for suppressing pro-inflammatory gene expressions in rheumatoid arthritis.