||Most leafy vegetables can accumulate large amounts of nitrate, which are often associated with harmful effects on human health. Nitrate assimilation in plants is determined by various growth conditions, especially light conditions including light intensity, light duration and light spectral composition. Red and blue light are the most important since both drive photosynthesis. Increasingly, recent evidence demonstrates a role for green light in the regulation of plant growth and development by regulating the expression of some specific genes. However, the effect of green light on nitrate assimilation has been underestimated. In this study, lettuce (Lactuca sativa L. cv. Butterhead) was treated with continuous light (CL) for 48 h by combined red and blue light-emitting diodes (LEDs) supplemented with or without green LED in an environment-controlled growth chamber. The results showed that nitrate reductase (NR) and nitrite reductase (NiR) related-gene expression and nitrate assimilation enzyme activities were affected by light spectral composition and light duration of CL. Adding green light to red and blue light promoted NR and NiR expressions at 24 h, subsequently, it reduced expression of these genes during CL. Compared with red and blue LEDs, green light supplementation significantly increased NR, NiR, glutamate synthase (GOGAT) and glutamine synthetase (GS) activities. Green-light supplementation under red and blue light was more efficient in promoting nutritional values by maintaining high net photosynthetic rates (Pn) and maximal photochemical efficiency (Fv/Fm).