The first detailed description of the proteome of the mouse jejunal brush border membrane vesicle is presented here. This was obtained by a combination of purification via divalent (Mg2+) cation precipitation starting with isolated cells plus strong cation exchange chromatography LC-MS/MS. Five-hundred seventy proteins were identified including 45 transport proteins. Among the latter, 18 had either not been identified in the intestine in the past or there was a single unconfirmed report of their presence. Validation was accomplished by a combination of immunoblotting and immunofluorescence using mouse jejunum and previously described antibodies. The validated BB proteins were aquaporin 7, Glut 9b, Na+I- symporter (NIS), and non-gastric H+/K+-ATPase. This study helps to further define the brush border membrane vesicle, a preparation which has been widely used to identify transport function of the small intestine.