Capparis spinosa L. “Caper” is one of the known medicinal plants in Palestine. In Arabic traditional medicine every part of Caper has medicinal uses, and roots particularly posses anti-inflammatory activity and commonly used for the treatment of rheumatism and back and joints pain. For better utilization of C. spinosa and to protect it from genetic erosion, a tissue culture protocol was optimized for the in vitro production of roots. MS media supplemented with IAA, IBA and NAA at different concentrations (0.0, 0.1, 0.5, and 1.0 mg/L) were evaluated for the production of adventitious roots under in vitro conditions. Results revealed that the highest rooting percentage (62.5%) was observed on when adding 1.0 mg/L NAA to the media. At 1.0 mg/L NAA, leaf discs from in vitro growing plants resulted in 68.8% rooting while leaves from ex vitro plants gave less percentage (43.8%). The effect of carbon type (sucrose, sorbitol and fructose) and concentration (1.5 or 3.0% w/v) on the in vitro adventitious root induction was tested. Sorbitol resulted in no rooting at both concentrations. Sucrose at 3.0% gave higher (41.6%) root induction compared to fructose (25.0%) at the same concentration. After six weeks from the induction, well established roots were transferred to 1X MS liquid medium containing 1.0 mg/L NAA and under continuous agitation at 80 rpm. The maximum root growth was obtained after six weeks of culture and later at the 6th week the growth was slowly declined. Key words: Capparis spinosa L., anti-inflammatory activity, root culture.