The focus of this study was to investigate the effect of light on the cultivation and the amounts of bio-active components in Flammulina velutipes. The mushrooms were cultivated under fluorescent tube (T8) grow lights, lightemitting diodes (LEDs), and cold-cathode fluorescent lamps. The biological efficiency of the T8 lights was the highest, at 92%. The crude fat content, crude fiber content, polysaccharide content, and ergosterol content were highest under the LEDs, at 2.9 g/100 g, 7.9 g/100 g, 3.9 g/100 g, and 1.4 mg/g, respectively. Moreover, vitamin D2 (1.9 μg/g) was generated only under light from LEDs. Principal component analysis showed that F. velutipes cultivated under the 3 different lighting conditions showed different profiles for proximate composition, nutritional compounds, and principal fatty acids.