This study investigated the induction and in vitro alkaloid yield of calluses and protocorm-like bodies (PLBs) from Pinellia ternata (Thunb.) Berit (Araceae). We planned to use this material in future studies related to the mass production of medicinally valuable compounds and regulation of alkaloid metabolism. Different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyladenine (6-BA), kinetin (Kin), and α-naphthaleneacetic acid (NAA) were used to induce callus and PLB formation from P. ternata tuber explants. The results showed that three physiologically distinct calluses were induced by different combinations of 2,4-D, 6-BA, and Kin used in this study. The calluses differed in color, texture, differentiation status, and alkaloid content. The alkaloid content of the three calli types ranged from 0.0175% to 0.0293%. In comparison, the alkaloid content of field-grown tubers was 0.0072%. Many reports have indicated that 2,4-D suppresses the biosynthesis of secondary metabolites; however, our results show that 2,4-D promoted alkaloid production in Pinellia calluses. The combination of NAA + 6-BA induced PLB formation. The PLB alkaloid content of 0.0321% was 1.1 to 1.8 times higher than the alkaloid content of the calluses and 4.5 times higher than the field-grown tubers. In conclusion, the induction of calluses and PLBs with alkaloid content greater than that of field-grown tubers indicates the potential use of these tissue culture materials for bioprocessing alkaloids from P. ternata and for the study of alkaloid metabolism.