A high-amylose rice with 64.8% amylose content (AC) was developed by transgenic inhibition of two isoforms of starch branching enzyme (SBE), SBEI and SBEIIb, in an indica rice cultivar. The expression of SBEI and SBEIIb was completely inhibited in the transgenic line, whereas the expression of granule-bound starch synthase was normal. Compared with wild-type rice, drastic reductions in both SBEs in the transgenic rice increased apparent AC in flour from 27.2% to 64.8%, resistant starch (RS) content from 0% to 14.6% and total dietary fibre (TDF) from 6.8% to 15.2%. Elevated AC increased the proportion of long unit chains in amylopectin and increased onset gelatinization temperature and resistance to alkaline digestion; however, kernel weight was decreased. A rat feeding trial indicated that consumption of high-amylose rice decreased body weight gain significantly (P < 0.01); increased faecal mass, faecal moisture and short-chain fatty acids; and lowered the faecal pH. An acute oral rice tolerance test revealed that the high-amylose rice had a positive effect on lowering the blood glucose response in diabetic Zucker fatty rats. This novel rice with its high AC, RS and TDF offers potential benefits for its use in foods and in industrial applications.