PAS kinase is a nutrient sensing serine/threonine protein kinase conserved from yeast to humans. Several previous studies have reported a variety of metabolic phenotypes associated with PAS kinase-deficient mice placed on a high fat (HF) diet including decreased weight gain and liver triglyceride accumulation, and increased metabolic rate, insulin production and insulin sensitivity. Herein we explore the role of gut microbiota in the PAS kinase-deficient(PASK-/-) mice on a western, high fat high sugar (HFHS) diet. Although the PASK(-/-) mice were not protected from weight gain or insulin insensitivity, they did display other markers of increased metabolic health including protection from liver triglyceride accumulation and decreases in the tight-junction protein claudin-1, which is associated with increased gut permeability and inflammation. PASK(-/-) mice did not display an alteration in the gut microbiome despite the observable expected increases in both Firmicutes and Actinobacteria on the HFHS diet. The HFHS diet did, however, result in a significant phenotypic split in a fraction of total mice on the HFHS diet, independent of genotype and including both WT and PASK(-/-) mice. Mice in the upper gut microbiota cluster displayed not only the altered gut microbiota, but decreased weight gain, whereas the mice in the lower gut microbiota cluster displayed decreased glucose sensitivity after both 19 and 23 weeks on the HFHS diet. The cause of the divergence in microbiota composition between the two clusters in unknown, however maternal influences are unlikely due to the similarity of the HFHS microbiota at 15 weeks. In addition, no gut microbiota differences or fractionation occurred in mice on the normal chow diet, suggesting a healthy diet protects against harmful disruptions to gut microbiota.