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Cnidium officinale Makino is an important medicinal plant of oriental clinics and is considered as the main source of phthalides, polyphenols, and flavonoids. However, there is no available report regarding the effect of different light colors on the secondary metabolites composition of C. officinale. In this study different light (dark, white, blue, red and red: blue) conditions were arranged to raise callus on MS medium containing 0.5 mg·L -1 of each 2,4-D and BAP. Callus grown in dark condition showed maximum (2.0 g) fresh weight with lower total phenolic and flavonoids contents. Also, in dark condition callus faced higher catalase (CAT) and guaiacol peroxidase (GPX) activities to avoid free radicals. Mix (red: blue) light condition favored the synthesis of phenolics and flavonoids in callus at the cost of higher ascorbate peroxidase (APX) and superoxide dismutase (SOD) enzymes expression. However, DPPH free radical scavenging activity was less variable among the samples from the different light conditions. Interestingly, the HPLC profile showed higher (28.3 μg·g -1 DW) phthalide accumulation in dark grown-cultures. Compared to other light conditions, 3-butyledinephthalide accumulation was higher (0.43 μg·g -1 DW) in white light-grown callus. These findings suggest that light conditions play an important role in the regulation of in vitro callus growth and synthesis of important medicinal compounds of C. officinale., (Copyright © 2019 Elsevier B.V. All rights reserved.)