Abstract: A rapid and high frequency reproducible in vitro regeneration protocol of a multipurpose bamboo species Dendrocalamus hamiltonii Nees et Arn. Ex Munro has been developed through single node cutting taken from the lateral branches of a 20 year old field grown elite bush. Axillary buds on the nodal explants sprouted within a fortnight of culture on Murashige and Skoog (MS) medium without any Plant Growth Substance (PGS). After 3-4 weeks of incubation, the sprouted buds were excised from the mother stumps and placed on MS medium supplemented with different concentrations of 6-benzylaminopurine (BAP; 2.0-12.0 μM) and 1.0 μM α-naphthaleneacetic acid (NAA). Enhanced proliferation was induced on the propagules (small clusters with 3-5 multiple shoots and rhizomatous part) on medium supplemented with 8.0 μM BAP and 1.0 μM NAA; subsequent removal of the shoots (about 1.5 cm) from the rhizomatous portion (shoot cut) and placing them on the same media combination influenced multiplication capacity. A multiplication of 20 folds was achieved on MS medium supplemented with 8.0 μM BAP and 1.0 μM NAA at the end of the 2nd subculture. Enhanced root formation (>90%) occurred when the propagules following shoot cut were placed on to MS medium supplemented with 100 μM indole-3-butyric acid (IBA) for 10 days and then transferred to IBA-free medium. This is the first report from this species where 20-fold multiplication was obtained and subsequent enhanced rooting (>90%). The hardened plants, established in the field, exhibited normal growth even after 2 years.