Abstract: Three-years-old apple trees (Malus domestica Borkh c.v.Rehd Fuji) were used as materials in this experiment. It was studied that the effect of Betaine (Bet), β- aminobutyric acid (β-ABA), Oligo-Chitosan (OC) and natural drought (ND) on leaf proline accumulation and water use efficiency (WUE) under controlling water treatments and natural water-losing of soil. The results show that β-ABA and OC increase proline content in secourd day with treatment and increas WUE on tenth day under RWC 70%～80%. The treatments of Bet, β-ABA, OC and ND all increase the content of proline in leaves during water-losing of soil. On fifth day of water-losing (RWC=50%～60%), leaf WUE is the highest in the plant treated by ND, in that by Bet secondly and OC thirdly. On tenth day of water-losing (RWC=50%～60%), leaf WUE decreas generally; the WUE of leaf pre-treated by Bet, β-ABA and OC all are higher than that by control significantly. Those results suggest that the pretreatments of Bet, β-ABA , OC and ND all can induce the accumulation of proline in a certain degree and resist the decrease of leaf WUE under severe water stress, in which effect of Bet is best and β-ABA secondly. Betaine can increase effectively the drought resistance and leaf WUE of apple tree.