Used a pool culture under rainproof, the effect of non-sufficient irrigation in different growth stages on leaf membrane lipid peroxidation, osmoregulation substances content and the protective enzyme activity of different peanut varieties were studied. The aim was to reveal the response mechanism of different peanut varieties to non-sufficient irrigation in different growth stages. The result showed that leaf protective enzyme activities, osmoregulation substances and malondialdehyde content reduced in different degrees after irrigation in seedling and flowering-pegging stages. Their activities increased with the growth of peanut and the reduction of soil moisture, but the increasing ranges were different because of peanut varieties, and kinds of protective enzymes or osmoregulation substances. The increasing range of leaf SOD, CAT, the content of soluble sugars, soluble proteins, free amino acids and proline of two varieties were larger in flowering-pegging stage, which is the most sensitive stage to water stress, than other stages. And the increasing range of leaf SOD, CAT, soluble proteins, and free amino acids content of Huayu 27 were larger than those of Huayu20. The protective enzymes activity and osmoregulation substances content were not reduced after irrigation in pod setting stage. The activity of protective enzymes and the osmoregulation substances content and MDA had no significantly difference between two varieties with irrigation in the whole growth stage and seedling stage. The activity of protective enzymes and the osmoregulation substances content and MDA in water stress under whole growth stage were increased significantly compared with irrigation treatment. The changing ranges of Huayu 27 were larger than those of Huayu22. The activity of POD was weakly affected by irrigation stage. SOD and CAT were the mostly protective enzymes of peanut to adapt to soil water stress.