Red and blue light have great effects on physiological processes and growth of plants. In this experiment, we investigated the physiological and growth response of pepper (Capsicum annuum L.) to supplementary red:blue (4:1) light for 1 h (T1), 3 h (T2), and 5 h (T3), and the full-spectrum light-emitting diodes, LEDs, as control (CK). Thirty-day-old seedlings were grown under these treatments for 20 days in a climate-controlled room before data measurement. The results showed that the light treatments significantly (p < 0.05) affected the photosynthesis and growth indexes as well as gene expression in the pepper seedlings. Plants under T2 generally had better performance in terms of seedling growth. A total of 124, 1283, and 1091 differentially expressed genes were found in CK vs. T1, CK vs. T2, and CK vs. T3, respectively. Among the treatments, T2 in comparison with CK had 705 upregulated and 578 downregulated differentially expressed genes (DEGs). We also found that CPRF2, Paggis, HLIPS, GIGANTEA, LSH1, and FTSH genes were expressed differently under the various light treatments. Based on GeneOntology (GO) enrichment analysis, DEGs were significantly enriched on 15 GO terms of which xyloglucan:xyloglucosyl transferase activity and apoplastic, cellular polysaccharide metabolic, and cellular carbohydrate metabolic processes were closely related to light responses. A total of 96 genes that are related to plant-pathogen interaction, zeatin biosynthesis, plant hormone signal transduction, and wax/cutin/suberine biosynthesis which are involved in the pathway of light reaction in plants were significantly enriched in T2 plants compared with plants under CK. The application of red:blue light at 4:1 for 3 h improved the growth of pepper seedlings better than the other treatments and this can be tested under the Chinese Solar Greenhouse condition.The exposure of 'Green Oak Leaf' lettuce plants to fluorescent lamps with blue or red LEDs increased stem diameter, leaf area, leaf number, and dry biomass of shoot [5]. Brown et al. indicated that red LEDs in combination with other wavelengths of light may be suitable for the culture of plants under climate-controlled environments [6]. The application of red and blue light also affected biomass accumulation, pigment synthesis, antioxidants, and phenolic compounds. Moreover, chlorophyll contents increased significantly with increasing blue light in tomato, cucumber, radish, and pepper as compared with control plants [7]. Košvancová-Zitová et al. reported that irradiance with high B:R ratio (3:1) increased the rate of photosynthesis in Fagus sylvatica better than irradiance with low B:R (1:3) ratio [8]. In another experiment, Son et al. reported that the total antioxidant phenolic concentrations of lettuce plants increased as the proportion of blue light was increased [9]. Furthermore, different proportions of red and blue light have different effects on plant growth. A study showed that photosynthesis and yield of plants treated with LED-A (R:B = 6:3) were improved as compared to...