Background: Arylnaphthalene lignan Justicidin B is a lead compound in the management of bone cancer and osteoclastogenesis . The compound is the main cytotoxic principle of rare medicinal plant Linum narbonense L. (Linaceae). However, there have been no reports on the bioreactor production of justicidin B. Objective: to develop cost-effective biotechnology for production of this anticancer metabolite. Materials and Methods: The genetic transformation in hairy roots induced by Agrobacterium rhizogenes strain ATCC 15834, was proven by PCR analysis. The control of bioreactor was synthesized by gradient method. The optimal values of the controlling parameters were estimated with presence of technological limitation. The general structure of control system was based on "Hardware in the Loop" (HIL). Results: Hairy roots produced five-fold higher yields of justicidin B (7.78mg/g DW) compared to callus. A rapidly growing root line was selected for cultivation in 2-L stirred tank bioreactor. After optimization, maximum biomass of 22.5 g.l -1 dry wt was harvested from the bioreactor culture vessel (recording about 8 times increase over initial inoculum), with 1.42 % ± 0.12 Justicidine B, greater than contents from flasks were obtained. The extracts were tested in a panel of human tumor cell lines, using the MTT-dye reduction assay, exert inhibitory effects against malignant cells. Conclusion: Our findings are the first work on large cultivation of L. narbonense hairy roots and bioreactor production of plant anticancer agent Justicidin B. To extend the research to human clinical studies, we have found a reliable biotechnological supply of plant material, produced this target compound.