The aim of this study was to establish an adventitious roots culture system for sterile plantlet segments of P. tunicoides and improved the accumulation of total saponins in cultured roots. Psammosilene tunicoides is a native Chinese plant with high commercial value as medicinal herb. Combination of NAA and IBA significantly affected the adventitious roots formation on agar-solided B_5 media and a maximal induction rate of 83% was obtained at 24±2°C with a photoperiod of 12 h. With a shaking of 110 rpm in darkness, transferring the detached adventitious roots to the growth regulator free 1/2 B_5 liquid media notably increased the biomass production compared to that on solid media over a 30-day-culture period. Further analyses showed that more saponins could be accumulated in the liquid culture than in the solid culture and the addition of exogenous oxalic acid to the liquid media could enhance the accumulation of total saponins in adventitious roots. These results suggested that adventitious roots culture will be an efficient alternative to the field cultivation of intact plants for the production of useful natural compounds from P. tunicoides.