Conjugated linoleic acid (CLA) has a role of biogenic regulation through modifying prostaglandin production. However, its effects on related metabolites of arachidonate remain unclear. Therefore, the effects of CLA on brain endocannabinoid content as well as its analogs were investigated. Mice (3-week-old), provided with diets containing 3% linoleic acid or 3% CLA for 4 weeks, were sacrificed and lipids were extracted from their cerebral cortex and hypothalamus. The amounts of N-arachidonoyl-ethanolamide, 2-arachidonoyl-glycerol (2-AG), oleoyl-ethanolamide and palmitoyl-ethanolamide were determined quantitatively by LC-MS. The 2-AG level in the cerebral cortex was significantly decreased by CLA treatment, but the other compounds were unaffected in the cerebral cortex and hypothalamus. The present study indicated that dietary CLA site-selectively decreases 2-AG in the cerebral cortex.