The desynchronization of circadian rhythms affected by light may induce physiological and psychological disequilibrium. We aimed to elucidate changes of growth, depression-anxiety like behaviors, melatonin and corticosterone (CORT) secretion, and gut microbiota in rats influenced by long-term light inputs. Thirty male Sprague-Dawley rats were exposed to a 16/8 h light/dark regime for 8 weeks. The light period was set to 13 h of daylight with artificial light (AL group, n = 10), or with natural light (NL group, n = 10), or with mixed artificial-natural light (ANL group, n = 10), and 3 h of artificial night light after sunset. The obtained findings indicated that the highest weight gain and food efficiency were observed in the AL group and the lowest in NL group. In the behavioral tests, the NL and ANL groups showed lower anxiety level than AL group, and ANL groups showed lower depression level than AL group. The NL and ANL groups had delayed acrophases and maintained higher concentrations of melatonin compared to AL group. The circadian rhythm of CORT was only found in ANL group. At the phylum level, the mixed light contributed to a lower abundance of Bacteroidetes. The genus level results recommend a synergistic effect of artificial light and natural light on Lactobacillus abundance and an antagonistic effect on the Lachnospiraceae_NK4A136_group abundance. The study indicated that the mixture of artificial and natural light as well as the alignment of the proportions had beneficial influences on depression-anxiety-like levels, melatonin and corticosterone secretion, and the composition of the gut microbiota. KEY POINTS: • The mixed light can reduce the depression-anxiety level • The mixed light can maintain the secretion rhythm of melatonin and CORT • The mixed light can increase Lactobacillus and decrease Lachnospiraceae_NK4A136_group.