Podophyllum peltatum is an important medicinal plant that produces podophyllotoxin (PTOX) with anti-cancer properties. We established the embryogenic cell and adventitious root culture systems in P. peltatum and analyzed PTOX production. For the growth of embryogenic cell clumps in shake flask culture, the most efficient concentration of 2,4-dichloroacetic acid (2,4-D) was 6.78 μM, and the growth of embryogenic cell clumps was 15.9-fold increased in Murashige and Skoog MS liquid medium with 6.78 μM 2,4-D after 3 wk of culture. To induce adventitious roots, half-strength MS medium showed the best results for adventitious root induction compared to full strength MS medium and MS medium lacking NH4NO3. Optimal indole-3-butyric acid concentration for adventitious root formation was 14.78 μM. In liquid medium, the frequency of adventitious root formation from root segments was 87.7% and the number of laterally formed adventitious roots was 22.3 per segment. PTOX production in embryogenic cells and adventitious roots was confirmed by liquid chromatography and electrospray ionization-tandem mass spectrometry analysis. High-performance liquid chromatography analysis revealed that adventitious roots contained higher PTOX than embryogenic cell clumps. Elicitor treatment (20 μM methyl jasmonate) strongly enhanced the production of PTOX in both embryogenic cell clumps and adventitious roots. This observation suggests that both embryogenic cell and adventitious root culture can be adopted to produce PTOX.