Abstract: In order to optimize the method of 2n pollen induction and to test the effects of colchicine on 2n pollen formation in nectarine, 2n pollen was induced by two colchicine treatments, cotton wool soaking and direct injection into floral buds of Prunus persica（L.）Batsch. var. nectarina（Ait.）Maxim. ‘Lichun’. Orthogonal experimental design was used in this study. In treatment of direct colchicine injection, 2n pollen ratio mounted to 85.0%, but fewer flowers developed. In treatment of cotton wool soaking, 2n pollen ratio was 44.1%, and the ratio of full developed flower was. The duration of treatment and humectant concentration were the key factors in cotton wool soaking. The maximum 2n pollen ratio was obtained when cotton wool soaking treatment lasted 32 hours and the 1% glycerol was used as humectant. Concerning to the ratio of flower harvested and 2n pollen percentage, the optimum method for 2n pollen induction in nectarine is 0.1%–0.2% colchicine treatment by cotton wool soaking for 24–32 h with 1 mL of laundry powder as saturation and 1% glycerol as humectant respectively.