Role of gut microbiome in obesity and type 2 diabetes (T2D) became apparent from several independent studies, indicating that gut microbiome modulators like prebiotics may improve microbiome perturbations (dysbiosis) to ameliorate metabolic derangements. We herein isolate water soluble, non-digestible polysaccharides from five plant based foods (acorn, quinoa, sunflower, pumpkin and sago seeds) and assess their impact on human fecal microbiome and amelioration of high fat diet (HFD)-induced obesity/T2D in mice. During polysaccharide isolation, purification, biochemical and digestion resistance characterization, and fermentation pattern by human fecal microbiome, we select acorn-and sago-derived prebiotics (on the basis of relatively higher purity and yield and lower protein contamination) and examine their effects in comparison to inulin. Prebiotics treatments in human fecal microbiome culture system not only preserve microbial diversity but also appear to foster beneficial bacteria and short-chain fatty acids (SCFAs). Feeding of acorn-and sago-derived prebiotics ameliorates HFD-induced glucose intolerance and insulin resistance in mice, with effects comparatively superior to those seen in inulin-fed mice. Feeding of both of novel prebiotics as well as inulin increases SCFAs levels in the mouse gut. Interestingly, gut hyperpermeability and mucosal inflammatory markers were *