Grain protein content （GPC） represents one of the critical factors affecting rice eating quality. It is of great significance for improving this trait through analyzing its genetic regulation mechanism. This study deployed a recombinant inbred line （RIL） population derived from two japonica rice cultivars， Zhengeng 2400 and Jiahe 218， showing significant difference on grain protein content. A quantitative trait loci（QTL）-Seq was used to locate the QTLs controlling grain protein content in rice. QTL mapping and fine mapping was performed using Ici Mapping 4.1 software. Through deploying △SNP index analysis of the F_8∶9 RIL population， four QTLs on chromosomes 1， 2， and 12 were identified. Eight QTLs on chromosomes 1， 2， 6， 7， 8， 9， and 11 in the F_9∶10 RIL population were detected by the Euclidean distance （ED） method analysis. A major QTL， designated qGPC1， accounting for 13.20% of the phenotypic variation with a LOD score of 3.91， was consistently detected both in F_8∶9 and F_9∶10 RIL. Fine mapping delimited qGPC1 to a 516 kb interval between markers 1-3782 and 1-3834. Sequence analysis of a reported GPC regulatory gene OsAAP6 revealed no polymorphisms between parents， suggesting the presence of a novel regulatory gene controlling GPC. A low-GPC line with superior eating quality from the RIL population was selected as a parental donor for developing the new cultivar strain Zhendao 1818. This advanced line exhibited significantly improved palatability scores to commercial cultivars Wuyunjing 23 and Nanjing 5055， along with increased plant height， seed setting rate， and thousand-grain weigh. Collectively， this study lays the foundation for further cloning of grain protein content genes and analyzing genetic regulatory mechanisms， and provides excellent germplasm resources for improving rice eating quality.