The chemical composition, anti-inflammatory, cytotoxic and antioxidant activities of essential oil from leaves of Mentha piperita (MEO) grown in China were investigated. Using GC-MS analysis, the chemical composition of MEO was characterized, showing that it was mainly composed of menthol, menthone and menthy acetate. MEO exhibited potent anti-inflammatory activities in a croton oil-induced mouse ear edema model. It could also effectively inhibit nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. The cytotoxic effect was assessed against four human cancer cells. MEO was found to be significantly active against human lung carcinoma SPC-A1, human leukemia K562 and human gastric cancer SGC-7901 cells, with an IC 50 value of 10.89, 16.16 and 38.76 µg/ml, respectively. In addition, MEO had moderate antioxidant activity. The results of this study may provide an experimental basis for further systematic research, rational development and clinical utilization of peppermint resources.