AbstractMushrooms possess antihyperglycemic effect on diabetic individuals due to their nonfibrous and fibrous bioactive compounds. This study aimed to reveal the effect of different types of mushrooms on plasma glucose level and gut microbiota composition in diabetic individuals. The effects of five different mushroom species (Ganoderma lucidum, GLM;Pleurotus ostreatus, POM;Pleurotus citrinopileatus, PCM;Lentinus edodes, LEM; orHypsizigus marmoreus, HMM) on alloxan‐induced diabetic rats were investigated in this study. The results indicated that LEM and HMM treatments showed lower plasma glucose levels. For the microbiota composition,ACE,Chao1,Shannon, andSimpsonwere significantly affected by PCM and LEM treatments (p <.05), while ACE, Shannon, and Simpson indexes were affected by HMM treatment (p <.01). Simpson index was affected in positive control (C+) and POM groups. All these four indices were lower in GLM treatment (p <.05). Dietary supplementation of mushrooms reduced plasma glucose level directly through mushrooms' bioactive compounds (agmatine, sphingosine, pyridoxine, linolenic, and alanine) and indirectly through stachyose (oligosaccharide) and gut microbiota modulation. In conclusion, LEM and HMM can be used as food additives to improve plasma glucose level and gut microbiome composition in diabetic individuals.