Large prospective cohort studies suggested an important role of cereal insoluble fiber in the prevention and management of type 2 diabetes, which challenge the traditional view that viscosity and solubility are the main driving factors for these beneficial effects of dietary fiber. To evaluate the anti-diabetic effects of insoluble- (BIF) and soluble fibers (BSF) enzymatically isolated from barley, a conventional rat model and a type 2 diabetes rat model were used. Our results showed that 4-week treatment of BIF or BSF effectively reduced FBG in the diabetic condition, with caecal level of propionic acid and minor SCFAs increased by BIF and that of butyric acid and insulin sensitivity improved by BSF, respectively. The two treatments further ameliorated liver function, judged by the recovered serum level of ALT, albumin and total protein levels. BIF and BSF also increased HDL-C and decreased serum MDA. In normal rats, BIF and BSF showed a hypolipidaemic effect in triglycerides and LDL-C, reduced body weight and enhanced the caecal production of minor SCFAs. Furthermore, the two treatments reduced the caecal level of butyric acid while BSF increased that of propionic acid. In conclusion, BIF could exert anti-diabetic effects that might via a different mechanism from BSF.