Swertia japonica Makino, often referred to as “Senburi” is used as traditional medicines in the treatment of gastrointestinal diseases in Japan. This plant is an important bitter stomachic that contains secoiridoids such as swertiamarin, gentiopicroside and those derivatives. We previously reported about the optimal media condition and chemical constituents of the callus, however the secoiridoids in the callus were extremely limited. To enhance the production of secoiridoids, in this study, we investigated the impact of light conditions on secoiridoid production under both solid and liquid culture conditions. Calli were incubated on the solid or liquid media with some light conditions adjusted with some different radiation intensities: 0 (dark) to 25.1 W/m2, or six different combinations of red, green, and blue (RGB): 2:3:1(white), 1:0:0(only red), 0:0:1(only blue), three purple light adjusted as 3:0:1, 1:0:1 and 1:0:3. After 4 weeks, methanol extracts of calli were analyzed by LC-ESI-MS. In the solid culture conditions, productions of swertiamarin and gentiopicroside were enhanced by increasing radiation intensities up to 18.1 W/m². Similar trends were observed under liquid culture conditions as well. Swertiamarin and gentiopicroside were the most produced in the calli cultured on the condition of white light compared with only red and blue light conditions in the solid culture. Moreover, the contents of swertiamarin and gentiopicroside were decreased by increasing the ratio of red light, suggested that red light might inhibit the production of swertiamarin and gentiopicroside. We have newly reported the effects of light conditions on secoiridoid production in this plant.