Abstract: 【Objective】Establish a micropropagation system for male and female of Ilex verticillata L. plant(Winterberry).【Method】In this work, we optimized the culture mediums of axillary bud induction, clustered bud proliferation and shoot rooting by using semi-lignied buds of male and female seedling and adding different regulators such as 6-benzylaminopurine(6-BA), indole-3-butyric acid(IBA), naphthaleneacetic acid(NAA), and N-(2-chloro-4-pyridyl)-N'-phenylurea(CPPU)in vitro.【Result】The optimal method for sterilization of explants was treatment with 70% alcohol for 30 s, and 0.1% HgCl₂ for 8 min; the optimal media for axillary bud induction of female and male explants were MS+0.5 mg/L 6-BA+0.2 mg/L IBA and MS + 0.5 mg/L 6-BA + 0.5 mg/L IBA, and this treatment resulted in axillary bud germination rates up to 93.3% and 83.3%, respectively. Furthermore, the optimal media for female and male multiple shoot clumps were MS + 1.0 mg/L CPPU + 0.2 mg/L NAA and MS + 1.0 mg/L CPPU + 0.1 mg/L NAA, this treatment resulted in proliferation rates up to 3.48 and 1.25, respectively. The shoots were induced to form roots on a 1/2MS + 0.4 mg/L NAA medium, and the rooting rates of females and males were only 26.7% and 20.0%, which increased to 66.7% and 46.7%, respectively, when a rooting agent was added to the medium. 【Conclusion】The establishment of this tissue culture system, especially the male plant tissue culture, provides technical support for the rapid propagation of Ilex verticillata species.