An efficient and rapid protocol for in vitro induction and complete plant regeneration of Polygonum multiflorum THUNB has been developed. Nodal explants were grown in vitro on Murashige and Skoog's (MS) basal medium containing different concentrations of alpha-naphthaleneacetic acid (NAA) and benzyladenine (BA). The nodal explants (97%) produced multiple shoots (4.7 shoots per explant) on MS basal medium supplemented with 0.2 mg/l NAA and 2.0 mg/l BA after 6 weeks of culture. Eighty-eight percent to 100% of the shoots (1.0 cm in length) elongated (about 3.02-4.28 cm) and rooted on MS basal medium supplemented with NAA or indole-3-butyric acid (IBA). All the rooted shoots were transferred to pots containing autoclaved soil, vermiculite, and peat moss (1 : 1 : 1). The plantlets were successfully acclimatized under greenhouse conditions with high humidity before transferring to the field. The anthraquinone contents were determined using HPLC. Analysis revealed that the contents of the major medicinal compounds-emodin and physcion in the 6 weeks old in vitro grown shoots and three month old in vitro propagated plants grown in greenhouse were higher than those of the marketed crude drug (processed underground or stem parts of P. multiflorum).