The purpose of present studywas to develop a micropropagation protocol of the important medicinal plant, Ocimum minimum L. from nodal stem explant. Nodal stem explants were inoculated on basal MS medium supplementedwith different concentrations of 6-benzylaminopurine (BAP), kinetin (KN) and combination of BAP with indole-3-acetic acid (IAA) for plant regeneration and 2,4-Dwere also used for callus induction.Maximum numbers of shoot (9.75±0.53) with highest length of shoots (5.95±0.08cm) were observed on the MS medium containing 1.0 mg/l BAP after 30-35 days of culture. Regenerated shoots were separated and rooted on half strengthMS medium supplemented with 1.0 mg/l of IBA alone for 25-28 days.Well-developed rooted plantlets were transferred to specially made plastic cup containing a mixture of garden soil, farm yard manure and sand (1:1:1) in controlled environment for longer duration for achieving high survival percentage (70-80%) for this plant. Acclimatized plantlets were successfully grown in garden soil. Thus, first time a comprehensive micropropagation protocol was developed for Ocimum minimum L. and it can be used for commercial propagation and in genetic improvement studies.