Sanqi, the root of Panax notoginseng, is a popularly used traditional Chinese medicine with cardiovascular effects. Notoginsengnosides (NG) isolated from Sanqi could inhibit ADP-induced platelet aggregation of rat washed platelets. To identify the possible target proteins of NG in platelets, two-dimensional gel electrophoresis (2-DE)-based comparative proteomics was performed and proteins altered in expressional level after NG treatment were identified by MALDI-TOF MS/MS. Treatment of 200 microg/ml NG caused regulation of the levels of 12 proteins, which play important roles in platelet activation, oxidative stress and cytoskeleton. In the NG-treated platelets, there were increase in the levels of growth factor receptor-bound protein 2 (Grb2), thrombospondin 1, tubulin alpha 6 and decrease in the levels of thioredoxin, Cu-Zn superoxide dismutase, DJ-1 protein, peroxiredoxin 3, thioredoxin-like protein 2, ribonuclease inhibitor, potassium channel subfamily V member 2, myosin regulatory light chain 9 and laminin receptor 1. The change in the levels of these proteins caused by NG treatment might contribute to the inhibitive effect of NG on platelet aggregation. Furthermore, analysis of the reactive oxygen species (ROS) level indicated that NG could decrease the ROS level in platelets. The regulation of ROS level might play important role in the effect of NG on platelets.