Environmental light has an important impact on the growth, development and oxidative stress of chicks. Thus, we investigated the effects of colored lights on microbes and explored the molecular mechanism by which external color light information alters the gut microbiota and induces the cell response in vivo. We raised 96 chicks under 400–700 nm white (WL), 660 nm red (RL), 560 nm green (GL) or 480 nm blue light (BL) for 42 days. We used 16S rRNA high-throughput pyrosequencing and gas chromatography to explore the effect of different monochromatic lights on the jejunal microbiota. We used qRT-PCR, western blotting, immunohistochemistry and Elisa to determine the effect of different monochromatic lights on small intestine development and oxidative stress levels. With consistency in the upregulation of antioxidant enzyme ability and anti-inflammatory cytokine level, the 16S rRNA and gas chromatography results showed that BL significantly increased the diversity and richness of the jejunal microbiota and improved the relative abundances of Faecalibacterium, Ruminiclostridium_9 and metabolite butyrate content compared with WL, RL and GL (p < 0.05). In addition, we observed that BL increased the goblet cell numbers, PCNA cell numbers, villus-length-to-crypt-depth (V/C) ratios, ZO-1, Occludin, and Claudin-1 protein expression; decreased permeability; and enhanced the digestion and absorption capacity in the jejunum (p < 0.05). In the in vitro experiment, we found that butyrate promoted chick small intestinal epithelial cell (CIEC) proliferation and inhibited apoptosis (p < 0.05). These responses were abrogated by the Gi inhibitor, PI3K inhibitor or AKT inhibitor, but were mimicked by GPR43 agonists or the GSK-3β inhibitor (p < 0.05). Overall, these findings suggested that BL increased the relative abundance of Faecalibacterium, Ruminiclostridium_9 and butyrate production. Butyrate may act as one of the signals to mediate blue-light-induced small intestinal development and mucosal barrier integrity enhancement and promote cell proliferation via the GPR43/Gi/PI3K/AKT/p-GSK-3β/β-catenin pathway.