The extraction and identification and anti-fatigue activity of iridoid glycosides in walnut meal were studied. The walnut was extracted by ethanol reflux, extracted with ethyl acetate and n-butanol. Different solvent extracts were used to determine the anti-fatigue effect by mouse running test and weight-bearing swimming test. The extract with clear anti-fatigue effect was further separated by MCI and ODS reverse-phase column chromatography, and the obtained monomer compound was identified by NMR. The results showed that the extract of n-butanol from the walnut meal extract had the most obvious effect on the anti-fatigue effect of the mice. The part of the ODS column was eluted with 5% ethanol to obtain the compound II. The compound was identified as the iridoid glycoside. And can significantly improve the anti-fatigue ability of mice.