A high-resistant starch (RS) and low-glutelin diet is beneficial for the health of patients with diabetes and kidney diseases. Rice is an important food crop worldwide. Previous studies have demonstrated that downregulating the expression of rice starch branching enzyme IIb (SBEIIb) affected the composition and the structure of starch. However, there has been no report about generating the loss-of-function mutants of SBEIIb using low-glutelin rice cultivars as recipients. In this study, we adopted a CRISPR/ Cas9 system to induce site-specific mutations at the SBEIIb locus in an elite low-glutelin japonica rice cultivar derived from Low Glutelin Content-1 (LGC-1) and successfully obtained two independent transgene-free sbeIIb/Lgc1 mutant lines. In the mutant lines, the apparent amylose content (AAC) was increased by approximately 1.8-fold and the RS content reached approximately 6%. The glutelin content was approximately 2%, maintaining the low-glutelin trait of the recipient cultivar. The formation mechanism of RS was explored by analyzing the fine structures and the properties of starch. According to the X-ray diffraction pattern and the increased lipid content, the high RS content of the sbeIIb/Lgc1 lines was attributed to the increased content of amylose−lipid complex. Further analyses of the nutritional quality revealed that the soluble sugar and lipid contents, especially sucrose and unsaturated fatty acids, increased in the sbeIIb/Lgc1 lines significantly. This research is expected to facilitate the cultivation and the application of functional rice suitable for patients with diabetes and kidney diseases.