Abstract: In the present study, anthers of Populus euphratica were used as explants to induce callus after disinfection and sterilization. The orthogonal design was used to explore the role of auxin and cytokinin in callus induction, followed by induction of callus differentiation and multiple shoots rooting. Finally, an efficient regeneration system of P. euphratica was established. The results show that the best method for sterilization as following: 70% alcohol soak for 30 seconds, then 8% sodium hypochlorite for 2 minutes, finally sterile water for 3 times; H medium supplemented with 1.0 mg/L NAA and 1.0 mg/L BA + 5.5 mg/L agar + 30 mg/L sucrose induced the highest rate of callus formation, the induction rate was 89%; MS medium + 1.0 mg/L NAA + 0.2 mg/L BA + 5.5 mg/L agar + 30 mg/L sucrose as the best medium for differentiation induction, the differentiation rate is about 50%; the rooting rate was the highest in 1/2 MS medium supplemented with 1.2 mg/L IBA + 5.5 mg/L agar + 20 mg/L sucrose, it can be reach 96.7%.