Grain protein content is one of the important factors affecting rice eating quality. It is of great significance for rice eating quality improvement to analyze the genetic regulation mechanism of grain protein content. Using an recombinant inbred lines (RILs) population from two japonica rice cultivars, Zhengeng 2400 and Jiahe 218, with significant different protein content of rice grain, BSA-Seq (bulked segregated sequencing) method was used to locate the QTLs controlling grain protein content in rice. Subsequently the QTL locus was further verified by QTL mapping using Ici Mapping 4.1 software and narrowed down by fine mapping. Four QTLs were found on chromosomes 1, 2, and 12 in F8:9 RIL by △SNP index method analysis, and eight QTLs distributed on chromosomes 1, 2, 6, 7, 8, 9, and 11 in F9:10 RIL were detected by ED method analysis. A major QTL locus designated as qGPC1 accounting for 13.2% of the phenotypic variation with a LOD score of 3.91, which was detected both in F8:9 and F9:10 RIL, was verified by ICIM and fine mapped. Finally, the qGPC1 locus was delimited in the region of 516 kb between markers 1-3782 and 1-3834. Of them, a reported gene OsAAP6 regulating grain protein content was no differences in sequencing between zhengeng 2400 and Jiahe 218. Maybe a new gene in this region regulates protein content of rice grain. Moreover, Zhendao 1818 with excellent eating quality and higher plant height, seed setting rate and thousand grain weight compared with the control variety was finally screened. 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.jian