We studied the effectiveness of different combinations of plant growth regulators and the light/dark condition on callus induction, proliferation and differentiation of bulb scales in Lilium martagon and established a high-frequency regeneration system. The optimal medium for callus induction was MS+0.2 mg?L^-1 TDZ+0.5 mg?L^-1 NAA, with embryogenic callus induced from 77.14% bulb scales. For callus proliferation, MS medium containing TDZ and NAA produced a browning appearance with the callus lost embryogenic ability quickly; therefore, 6-BA was applied instead of TDZ. Generation of buds from callus was reduced with 6-BA concentration increasing, which indicates that 6-BA plays a key role in keeping meristematic cells of callus from differentiating. The optimal medium for callus proliferation was MS+0.5 mg?L^-1 6-BA+0.1 mg?L^-1 NAA, with the proliferation coefficient 2.93. Dark treatment led to the highest callus induction rate, proliferation coefficient and number of buds/callus (77.14%, 2.93 and 5.43, respectively); callus showed good growth and the buds could grow roots and develop complete plants, which suggests that the dark condition is more appropriate for in vitro regeneration than the light condition. The rapid propagation system of L. martagon established in this study offers insights into germplasm protection, genetic transformation and application of L. martagon in China.