Abstract: The aims were to study the effects of different culture conditions on regeneration status of adventitious bud and rooting rate of Hydrocotyle vulgaris L., and screen the optimal culture condition. Using auxiliary stems (about 1.0 cm) of Hydrocotyle vulgaris L. as explants, adventitious bud induction, multiple shoots proliferation and plant regeneration were the focal points in the research. The results showed that type and mass concentration of growth regulator had obvious effects on sprouting time and regeneration rate of adventitious bud. The proliferation rate was 89%, and the rooting rate was 98%. The culture condition was illumination intensity 1500 lx, illumination time 12 h/d and culture temperature 25℃. The effective medium for cluster buds-inducing was MS 6-BA 0.12 mg/L NAA 0.3 mg/L. The effective medium for rooting rate was MS 6-BA 0.3 mg/L NAA 0.15 mg/L.