A regeneration system from protoplast to plantlet for a medicinal plant species, Phellodendron amurense Rupr., has been developed. Leaves of micropropagated shoots or plantlets were selected as plant materials for protoplast isolation. The yield and viability of leaf protoplasts were greatly influenced by the enzyme combination, treatment time and osmoticum. The highest viability (86%) with a yield of 7.1×10⁵ protoplasts per gram fresh weight was obtained with a 6-h digestion in 1% Cellulase Onozuka R-10 plus 1% Driselase-20. Sustained cell division and colony formation from the protoplasts were best supported at a plating density of 4×10⁵–6×10⁵ protoplasts per milliliter using a 0.2% gellan gum-solidified or liquid MS (Murashige and Skoog, 1962) medium containing 0.6 M mannitol, 2.0 μM 6-benzylaminopurine (BA) with 4.0 μM α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or 2,4-dichlorophenoxyacetic acid (2,4-D). The protoplast-derived colonies formed green compact calluses when transferred to a solidified MS medium containing 2.0 μM BA with 4.0 μM NAA or IBA. Shoot regeneration from protoplast-derived calluses was induced on MS medium supplemented with 2.0 μM BA and 1.0 μM NAA or 2.5 μM IBA. Shoot multiplication and elongation occurred on MS medium containing 1.0 μM BA. In vitro-grown shoots were rooted on MS medium with either 0.5–4.0 μM IBA or NAA. Regenerants were transferred to the Kanuma soil and successfully established under greenhouse conditions.