The study was conducted to establish callus culture and plant regeneration in Withania somnifera (L.) Dunal. cv. Poshita with different explants and different auxin types and concentrations. Nodal explant showed better callusing response irrespective of auxin concentrations supplemented in the media, as compared to epicotyls, hypocotyls, leaves, and root tips. Maximum callusing score of 4.28 was recorded when nodes were cultured on Murashige and Skoog’s (MS) media containing 2 mg · L−1 naphthaleneacetic acid (NAA). Root tip and hypocotyls explants, which were found poor in inducing callus, showed highest proliferation of 6.57 and 6.00, respectively, on media with 2,4-Dichlorophenoxyacetic acid (2,4-D) for subculture. Highest number of shoots (16.0) per explant was obtained from epicotyls and nodes cultured on 1 and 2 mg · L−1 benzyladenine (BA), respectively. Indole-3-butyric acid (IBA) was found to be superior source of auxin for all rooting parameters, except percent rooting. Number of primary roots was found maximum (24.8) with 2.0 mg · L−1 IBA. Irrespective of auxin used, the length of primary root decreased with increase in concentration, maximum (6.18 cm) being in media with 0.5 mg · L−1 IBA. Plantlets were best hardened when agropeat was used as a substrate.