Lithospermum erythrorhyzon has been used as a red fooddye and traditional Chinese medicine to treat wounds, skin diseases and burns. Platelet activation plays an important role in thrombosis and haemostasis. Here, we studied the inhibition of platelet activation and its active compound from the root of Lithospermum erythrorhyzon. Its ethyl acetate extract inhibited the aggregation of washed rabbit platelets induced by collagen or thrombin. Five naphthoquinone pigments, shikonin, acetylshikonin, isobutylshikonin, α-methyl-n-butylshikonin and (β,β-dimethylacrylshikonin were isolated by means of high pressure liquid chromatography. The structures were determined by comparison of their proton nuclear magnetic resonance spectra. The potency of their inhibition was in the following order : β,β-dimethylacrylshikonin≥α-methyl-n-butylshikonin$gt;isobutylshikonin$gt;acetylshikonin$gt;shikonin. It is suggested that the size of the aliphatic hydroxy group of shikonin is important for the enhancement of potency.