Our study demonstrated that sleep deprivation resulted in homeostasis disorder of colon. Our study goes deeper into the positive effects of melatonin on small intestinal microbiota disorder caused by sleep deprivation. We successfully established a multiplatform 72 h sleep deprivation mouse model with or without melatonin supplementation, and analyzed the change of small intestinal microbiota using high-throughput sequencing of the 16S rRNA. We found melatonin supplementation suppressed the decrease of plasma melatonin level in sleep deprivation mice. Meanwhile, melatonin supplementation improved significantly the reduction in OTU numbers and the diversity and richness of jejunal microbiota and the abundance of Bacteroidaeae and Prevotellaceae, as well as an increase in the Firmicutes-to-Bacteroidetes ratio and the content of Moraxellaceae and Aeromonadaceae in the jejunum of sleep deprived-mice. Moreover, melatonin supplementation reversed the change of metabolic pathway in sleep deprived-mice, including metabolism, signal transduction mechanisms and transcription etc, which were related to intestinal health. Furthermore, melatonin supplementation inverted the sleep deprivation-induced a decline of anti-inflammatory cytokines (IL-22) and an increase of the ROS and proinflammatory cytokines (IL-17) in jejunum. These findings suggested that melatonin, similar to a probiotics agent, can reverse sleep deprivation-induced small intestinal microbiota disorder by suppressing oxidative stress and inflammation response.