Peganum harmala L. is a shrub perennial plant of Zigophyllaceae. It is used for treatment of parkinson in folk medicine and has antitumor and antioxidant activity. This species is usually propagated by seeds but the span of seed viability is short. On the other hand, since this is a wild plant and is not cultivated, it is exposed to extinction due to overuse. In order to solve this problem and optimize secondary metabolite production in this plant, the first step is in-vitro optimization of callus induction and shoot regeneration. To achieve this goal, leaf, hypocotyl and embryo axis were cultured on MS medium containing different concentrations of 2,4-D (0, 0.25 and 0.5 mg L-1) in combination with BA (0, 0.5, 1 and 2 mg L-1). The results of analysis of variance showed that the main effects of hormones and explants and some interactions were significant on callus induction and shoot regeneration. Maximum callus induction and shoot regeneration was obtained in the medium supplemented with 0.5 mg L-1 2,4-D and 1 mg L-1 BA for the leaf and hypocotyl explants, respectively.