Rosa rugosa Thunb, a deciduous shrub of the genus Rosa, has been widely used to treat stomach aches, diarrhoea, pain, and chronic inflammatory disease in eastern Asia. In recent years, our research team has extensively studied the Rosa rugosa flower extract, and specifically undertook pharmacological experiments which have optimized the extraction process. Our methods have yielded a standard extract enriched in phenolic compounds, named PRE. Herein, we expand our efforts and evaluated the anti-inflammatory activity of PRE on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages. PRE significantly inhibited production of nitric oxide (NO), prostaglandin E-2 (PGE(2)), tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and interleukin 1 beta (IL-1 beta), as well as expression of their synthesizing enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase2 (COX-2). Furthermore, PRE inhibited activity of mitogen-activated protein kinases (MAPK) as well as nuclear factor-kappa B (NF-kappa B) signaling pathway. Our findings are the first to explain the anti-inflammatory mechanism by PRE in LPS-stimulated macrophages. Given these results, we propose that PRE has therapeutic potential in the prevention of inflammatory disorders.