An efficient, rapid, and highly reproducible regeneration protocol was successfully developed for Phyllanthus fraternus from the field-derived mature nodal segments. The explants induced multiple shoots on cytokinin containing medium. The highest frequency (99 %) and maximum number of shoots (19.75) were induced on Murashige and Skoog's (MS) medium supplemented with 2.22 mu M 6-benzylaminopurine after 3-4 weeks of culture initiation. The elongated shoots were rooted on MS medium supplemented with indol-3-butyric acid (IBA) or alpha-naphthalene acetic acid. Pulse treatment of microshoots promoted significant increase in the percentage of rooting and number of root regeneration per shoot. The highest rooting (100 %) and maximum number of roots (8.75) per shoot was obtained when shoots were dipped in IBA solution (0.98 mM) for 5 min and further subcultured on MS basal medium. Plantlets were successfully acclimatized and established in soil. Regenerated plants were grown normally in the field without showing any morphological variations. This cost-effective protocol will help the mass multiplication of P. fraternus for commercial propagation and high biomass production of this valuable medicinal plant.