Vasoactive intestinal peptide (VIP) regulates clock gene expression in the brain that synchronizes diurnal feeding behaviors in mammals. In the gastrointestinal (GI) tissues, VIP influences host nutrient absorption from ingested food, and regulates host metabolic functions. VIP signaling ensures efficient nutrient absorption by influencing ghrelin and leptin expression to balance caloric intake. Importantly, obese humans have elevated plasma VIP levels, supporting its association with fat mass accumulation. In contrast, VIP deficiency leads to weight loss and reduced adiposity, while disrupting epithelial cell nutrient absorption, tight junctions and mucus secretion. Moreover, VIP regulates host glucose metabolism as VIP knockout mice are pre-diabetic with elevated blood glucose and insulin levels. In addition to metabolism, VIP is anti-inflammatory and when knocked out results in exacerbated inflammatory bowel disease (IBD) pathology. The GI track is also home to ≈40 trillion bacteria, called the gut microbiota, which unlock additional calories from fiber for the host. Microbiota dysbiosis is caused by dysfunction in biological systems downstream from VIP signaling, including dysregulated expression of host clock genes, metabolic hormones, immune-relevant mediators and metabolic and inflammatory disease states, like obesity and IBD. It is not known, however, whether the VIP signaling axis contributes to the maintenance of the gut microbiota structure and diversity. We hypothesized that VIP deficiency will cause gut dysbiosis, lower bacterial diversity and reduce its energy extraction potential. To this end, we isolated fecal samples from VIP knockout mice (VIP-/-) and employed 16S rRNA sequencing. VIP deficiency (VIP-/- and VIP-/+) resulted in marked gut microbial compositional changes and reduced bacterial diversity compared to male and female VIP+/+ littermates (n=48). Increased abundance of Bacteroides, Parabacteroides and Helicobacter genera (gram-negative, GN), with reductions of Lachnospiraceae NK4A136, Oscillibacter and Ruminiclostridium genera (gram-positive, GP), were the driving force for the observed increase in the GN/GP ratio. A predicted algorithm program, called PICRUSt, showed changes in microbial metabolism consistent with elevated lipopolysaccharide metabolism and reduced intake of fiber in VIP-/- mice. These data support that VIP regulates intestinal homeostasis by maintaining microbiota balance, diversity and energy harvesting potential, while upholding an anti-inflammatory tone by limiting lipopolysaccharide biosynthesis.