Cordyceps militaris (L.) Link (C. militaris) has been used as a folk medicine for treatment of various diseases in China and some other countries. Recent evidence suggests that aqueous extracts of C. militaris have hypoglycemic activity. So the aim of this study was to isolate and characterize compounds with aiti-PTP1B (protein tyrosine phosphatase 1B) activity from C. militaris. As a result, cordycerebroside B (1) together with other three known cerebrosides (2-4) and a disaccharide (5) were isolated by silica gel column chromatography and semi-preparative high performance liquid chromatography (HPLC) and then elucidated on the basis of 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, mass spectroscopy (MS) and chemical method. Among of which, cordycerebroside B was a new compound and isolated from C. militaris for the first time. The results of the activity assays demonstrated that all these four cerebrosides (compounds 1-4) showed marked inhibition activity against PTP1B with IC