Boerhaavia diffusa Linn. Belonging to family Nyctaginaceae is an important medicinal plant. An efficient protocol was developed for in vitro propagation of B. diffusa. Leaf explants was used for callus induction and regeneration of B. diffusa on MS basal medium, with different concentrations and combinations of cytokinins (BAP, Kinetin) and auxins (NAA, IAA). Best response to shoot proliferation was achieved with MS medium supplemented with 3.0mg/l BAP and 1.0mg/l NAA. Efficient rooting was achieved at low concentrations, 0.5mg/l of IAA. About 80% of the regenerated plants survived in the field conditions.