As we previously reported, cAMP and p38 MAPK instead of protein kinase A were involved in beta-adrenergic receptor (beta-AR)-mediated interleukin-6 (IL-6) production in mouse cardiac fibroblasts. Besides kinases, phosphatases may also be involved in IL-6 gene regulation. To study the role of protein tyrosine phosphatases (PTPs) in beta-AR-mediated IL-6 production, we selected the most widely used PTP inhibitor, phenylarsine oxide (PAO). We found that PAO dose-dependently inhibited the IL-6 release in response to beta-AR agonist isoproterenol (ISO) in mouse cardiac fibroblasts. This effect was probably due to the inhibition of PTPs, resulting in increased tyrosine phosphorylation, since genistein, an inhibitor of protein tyrosine kinases further potentiated ISO-induced IL-6 production and could partially reverse the inhibitory effect of PAO. PAO also significantly inhibited the IL-6 production by forskolin, an adenylyl cyclase (AC) activator. Furthermore, PAO dose-dependently inhibited the increased cAMP accumulation by either ISO or forskolin and suppressed the phosphorylation of CREB, an important transcriptional factor for IL-6 gene expression. But PAO did not affect the activation of p38 MAPK by ISO. Although PAO was also reported to inhibit NADPH oxidase, the inhibition of NADPH oxidase by its specific inhibitor, diphenylene iodonium (DPI) could not suppress beta-AR-mediated IL-6 production, suggesting that NADPH oxidase may not contribute to the inhibitory effect of PAO on IL-6 production. To our knowledge, this is the first report that PAO can inhibit ISO-induced IL-6 expression and CREB phosphorylation, demonstrating that PTPs may negatively regulate beta-AR-mediated IL-6 production. This study may also further our understanding of beta-AR signaling and provide potential therapeutic targets for the treatment of heart diseases.