Simon poplar (Populus simonii) protoplasts were isolated from suspension cells, with protoplast yield of 3.8×10(7) g(-1) F. W. They were cultured in a K8P liquid medium containing 13.57μM 2,4-D, 1.07μM NAA and 0.93 μM KT. Protoplast culture was influenced by the plating density, osmotic pressure, and the sources and amounts of nitrogen and carbon in the culture medium. Multiple shoots were produced from protoplast-derived callus after culture on MS medium containing 4.44 μM BA, 2.32μM KT, 2.28 μM ZT, and 0.54μM NAA. Shoots 2-3 cm in height were isolated from the calli and rooted on 1/2 MS medium. After transplantation into pots, the regenerated plants grew vigorously in greenhouse.