The breeding of functional rice varieties with high resistant starch content has now become an important direction in hybrid rice breeding. SBE3 (Starch Branching Enzyme 3), a key functional gene in the resistant starch biosynthetic pathway, encodes a starch branching enzyme. In this study, the SBE3 gene in Huazhan, an elite indica rice restorer line with high eating quality and resistance to rice blast, was edited and knocked out using CRISPR-Cas9-mediated technology. Three T0 transgenic lines carrying homozygous mutation at the locus of SBE3 were generated, and 60 T1 individuals derived from these three plants were genotyped by PCR amplification and sequencing analysis. Two homozygous mutant lines, named HZ-sbe3-rs1 and HZ-sbe3-rs2, which had deletions of two and seven nucleotides, respectively, in the third exon of SBE3 and were free of exogenous vectors, were finally obtained. The resistant starch content was dramatically increased in HZ-sbe3-rs1 and HZ-sbe3-rs2. Concomitantly, starch granule morphology and ultrastructural development were abnormal in both mutant lines. RT-PCR and qRT-PCR revealed that SBE3 gene is specifically expressed in young panicles during the filling stage in rice. Compared with the wild-type Huazhan, the mRNA transcript level of the SBE3 gene was markedly lower in HZ-sbe3-rs1 and HZ-sbe3-rs2. DNA fingerprinting analysis showed that HZ-sbe3-rs1 and HZ-sbe3-rs2 exhibited an extremely high genetic similarity with wild-type Huazhan at the DNA level. Phenotypic and agronomic characteristics evaluation indicated that the key yield-related traits of HZ-sbe3-rs1 and HZ-sbe3-rs2 did not differ significantly from those of the wild type. Further investigation demonstrated that SBE3 plays a vital role in regulating the expression of starch synthesis-related genes in the endosperm during the grain-filling stage in rice. Loss-of-function mutations in SBE3 will seriously disrupt the transcriptional expression of these genes at the RNA level, thereby altering the content of amylose and resistant starch in mature grains and, to some extent, affecting the grain appearance quality, cooking and eating quality of rice. By crossing the CMS (cytoplasmic male sterile) line Tianfeng A with HZ-sbe3-rs1 and HZ-sbe3-rs2, respectively, two hybrid combinations Tianfeng A/HZ-sbe3-rs1 and Tianfeng A/HZ-sbe3-rs2 were generated. Of which, the key agronomic characteristics and yields were comparable to those of the control variety Tianyouhuazhan. Therefore, CRISPR/Cas9 technology can be used to edit the SBE3 gene and improve the resistant starch content in rice. It provides theoretical guidance for development of novel indica-rice restorer line with excellent comprehensive traits and significantly increased resistant starch content, leading to an accelerated breeding process of functional hybrid rice with high resistant starch content.