Irisflorentin, a naturally occurring isoflavone, is an abundant active constituent in Rhizoma Belamcandae. Although some chemical studies have been reported, pharmacological actions of irisflorentin are not well studied. In this study, we demonstrate the anti-inflammatory activity of irisflorentin in lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages. Irisflorentin markedly reduces the transcriptional and translational levels of inducible nitric oxide synthase (iNOS) as well as the production of NO. Furthermore, it also significantly inhibits TNF-α, IL-1β and IL-6 at both the transcriptional and translational levels. These effects mainly act via ERK1/2 - and p38-mediated the activator protein-1 (AP-1) rather than the nuclear factor-κB (NF-κB) pathway. Thus, our study elucidates the anti-inflammatory mechanism of irisflorentin in LPS-activated RAW 264.7 macrophages.