A procedure for plant regeneration from protocallus of Radix Gentianae Macrophyllae was developed. The protoplasts were isolated from callus derived from tender leaves, using an enzyme solution containing 2% Cellulase, 1% Hemicellulose, 0.5% pectolyase, 0.05mol/L CaCl 2 , 0.4 mol/L mannitol and 0.1% 2[N-morpholino] ethanesulfolic acid (MES). The highest yield of protoplasts (4.23×106/g FW) was obtained from callus of subculture 12 d. The viability of protoplasts was up to 80%. The protoplasts sustained divisions were obtained in DPD medium supplemented with 1.5 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.5 mg/L 6-benzylaminopurine (6-BA), 0.3 mol/L mannitol, 2% sucrose and 500 mg/L casein hydrolysate (CH) at the plating density of 4.0×105 /ml. Protocallus formed a large amount of embryoids in MS medium supplemented with 0.2 mg/L 2,4-D, 1.2 mg/L 6-BA, 3% sucrose and 0.65% agar. The differentiation frequency from protocallus reached to over 96.55%.