Abstract: Leaf and internode explants of Withania coagulans Dunal were used to evaluate the effect of growth regulators on the in vitro callus induction and plant regeneration. Two independent experiments were performed to establish callus and plant regeneration. In the first experiment, leaf segments were cultured on MS basal medium fortified with 2, 4-dichlorophenoxyacetic acid (2, 4-D, 2-4 mg L-1) with combination of 6-benzyladenine (BA, 0.5-1 mg L-1) or kinetin (Kin, 0.5-1 mg L-1). Callus initiation was observed best in all media after 14-16 days (100%). Highest callus growth in terms of dry weight (76±5.34 mg) was observed in MS medium fortified with 2 mg L-1 2, 4-D and 0.5 mg L-1 Kin. In the second experiment, callusing also obtained from internode segments cultured on MS medium with the presence of 2, 4-D (2-4 mg L-1) in association with BA (0.25-0.5 mg L-1). Best callusing rates (42%) and dry weight (86±3.68 mg) occurred in the presence of 4 mg L-1 2, 4-D with combination of 0.25 mg L-1 BA. Shoot induction was obtained just from callus induced from internode explants on MS medium containing BA (2 mg L-1) with IBA (0.5 mg L-1), but the yield of shoot regeneration was unsatisfactory (18-33%). Regenerated shoots were rooted best (100%) on half strength MS medium containing IBA (2 mg L-1) and produced 23 roots with an average root length of 6.5±0.41 cm. Rooted plantlets transferred to soil and sand mixture (2:1) showed 75% survival when transferred to outdoor. The procedure reported here in vitro culture of W. coagulans for first time.