Abstract: Aconitum balfourii Stapf is a rare medicinal herb belonging to genus Aconitum and family Ranunculaceae. Excessive illegal collection from wild due to pharmacological significance along with quiescent nature of seeds and lengthy cultivation cycle are collectively responsible for its endangered status. Therefore, conservation applying tissue culture is a vital need and has been developed nowadays. In the present study, in vitro regeneration of A. balfourii from root explants has been attempted. Root explants were cultured on MS-Medium supplemented with different combinations of BA and NAA and callus induction took place at 13.4 μM NAA and 5.55 μM BA. Such induced calluses were transferred to shooting media. The best shooting were observed in 8.88 μM BA and 0.54 μM NAA. These shoots were subculture on 7.99 μM BAP. Furthermore the result showed that increasing the concentration of BA caused a reducing length of shoots; also presence of low concentration of NAA has been necessary for shoot induction. Some factors such as shoot length, no. of shoots and induction frequencies of shooting and rooting were also studied. The well developed micro shoots were cultured on rooting media for complete regeneration of plant. Rooting of micro shoots occurred in media containing 1.43 μM IAA and 1.23 μM IBA.