PNU-120596 is a classical positive allosteric modulator (PAM) of alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) and widely used to investigate the effect of alpha7 nAChR activation on several inflammation-associated diseases including rheumatoid arthritis, inflammatory bowel disease and cerebral ischemia. In this study, we report that PNU-120596 directly inhibits p38 mitogen-activated protein kinase (MAPK) activity. In 293A cells, p38 MAPK phosphorylation by several factors (oxidative stress, osmotic stress, TNF-alpha, or muscarinic stimulation) was inhibited by PNU-120596 as well as p38 MAPK inhibitor BIRB-796. Inhibition of p38 MAPK phosphorylation by PNU-120596 was not affected by alpha7 nAChR antagonist, methyllycaconitine (MLA). In vitro kinase assay revealed that PNU-120596 directly inhibits p38alpha MAPK-induced activating transcription factor 2 (ATF2) phosphorylation. MKK6-induced phosphorylation of p38alpha MAPK was also inhibited by PNU-120596. Real-time monitoring of binding to p38alpha MAPK using fluoroprobe SKF-86002 showed quite rapid binding of PNU-120596 compared to BIRB-796 which is known as a slow binder. Finally, we showed that PNU-120596 suppressed LPS-induced phosphorylation of p38 MAPK and expression of inflammatory factors including TNF-alpha, IL-6 and COX-2, independent on alpha7 nAChR activity in microglial cell BV-2. Thus, PNU-120596 might exert an anti-inflammatory effect through not only alpha7 nAChR potentiation but also direct inhibition of p38 MAPK.