Functional oligosaccharides show anti-diabetic effects through inflammation regulation with improved glucose metabolism. In this study, novel prebiotics of manno-oligosaccharides from cassia seed gum (CMOS) were incorporated into the diet of streptozotocin (STZ) plus high-fat and high-sugar diet (HFSD)-induced rats. After feeding for 8 weeks, CMOS (300-1200 mg per kg b.w. per d) significantly ameliorated the fasting blood glucose level (7.1-8.2 mmol L-1) as compared with that of the model group (14.2 mmol L-1), where the area under the oral glucose tolerance test curve was decreased by 20.0%-24.5%. Meanwhile, CMOS prevented STZ plus HFSD-induced damage to islet tissue with a clear and integrated morphology and reduced the glucagon/insulin area ratio (by 97.9% for 300 mg per kg b.w. per d CMOS). CMOS also reduced metabolic endotoxemia and maintained intestinal integrity with recovered mRNA expression of Zo-1 and occludin to the normal comparable level. Upon 16S rDNA sequencing, it was found that CMOS regulated the microbiota composition in the cecum with an increased relative abundance of Bifidobacteria, while that of Shigella was decreased. The molecular mechanisms involved in the anti-diabetic effects of CMOS were further studied. CMOS reduced the mRNA expression of Tlr2 and Tlr4 in the intestines of STZ plus HFSD-induced rats. Meanwhile, Nlrp3 associated inflammasome activation in the intestine and liver with glucose metabolism disorder was inhibited by CMOS, resulting in reduced interleukin-1β secretion (by 38.8-46.4% for CMOS of 300-1200 mg per kg b.w. per d) and inflammation. Furthermore, CMOS regulated the AKT/IRS/AMPK signaling pathway and improved glucose metabolism in the liver. Findings obtained here implicated that CMOS could modulate metabolic-inflammation as a functional dietary supplement.