Abstract: This paper reported that the cotyledonary generated from somatic embryos of Aesculus hippocastanum L. can be used to induce somatic embryogenesis in vitro culture repetitively. The results showed that the secondary somatic embryos reinduced from cotyledon directly, which was cultured on MS medium supplemented with ZT(0.5～2.0mg/L)+BA (1.0mg/L)+IAA (0.2mg/L). The induction rate was over 80%. But, the differentiation ability of the different parts of the same cotyledon was totally different, the closer to the hypocotyl, the better differentiation ability was.