Plumbagin is as an important bioactive secondary metabolite found in the roots of spp. The only one species, L., grows wild in Iran. The therapeutic use of plumbagin is limited due to its insufficient supply from the natural sources as the plants grow slowly and take several years to produce quality roots.To develop an efficient protocol for the establishment of callus and cell suspension cultures of and to evaluate production of plumbagin in callus and cell suspension cultures of for the first time.Stems and leaves explants were cultured on agar solidified (7% w/v) MS media, supplemented with different combination of 2, 4-D and Kin or 6-Benzylaminopurin (BA) for callus induction. The rapid growing calli were cultured in liquid Murashige and Skoog (MS) media in agitated condition for establishing cell suspension cultures of . Moreover, the effects of light and dark conditions on the cell growth, cell viability and plumbagin production in cell suspension cultures of were assessed.Friable calli were successfully induced using stem segments of in semisolid MS medium supplemented with 1 mg.L 2, 4-Dichlorophenoxy acetic acid (2, 4-D) and 0.5 mg.Lof kinetin (Kin). Optimal cell growth was obtained when the cells were grown in MS liquid media supplemented with 1 mg.L 2, 4-D and 0.5 mg.L kinetin with an initial cell density of ~3×10 cells per ml incubated in the dark at 25 ± 1 °C. Growth curve revealed that the maximum cell growth rate (14.83×10 cells per ml) achieved on the day 18 and the highest plumbagin content (0.9 mg.g Dry Cell Weight (DCW)) in the cells was obtained at the late exponential phase under dark condition which determined by High Performance Liquid Chromatography (HPLC) technique. Based on the obtained results, cell viability remained around 82.73% during the 18 days of cell culture in darkness. These suspension cultures showed continuous and stable production of plumbagin.Our study suggests that cell suspension cultures of represent an effective system for biosynthesis and production of plumbagin as a valuable bioactive compound.