Abstract: Grain protein content (GPC) is well-known to negatively influence the eating and cooking quality in rice. However, the genetic mechanism underlying the natural variation of GPC is very limited. Here, we identify GPC6 as a key determinant through genome–wide association analysis. GPC6 encodes a protein with a MYB–like domain, functioning as a transcriptional repressor to modulate the expression of storage protein synthesis genes (Alb1, Pro13b.5, RM1, OsGluB1b, OsGluB7) and two regulators (RISBZ1 and PDIL1–1). Further, one functional SNP (-1,471 T/C) in the GPC6 promoter was identified, which defines two haplotypes: high–expression GPC6^T (Low protein) and low–expression GPC6^C (High protein). Notably, the GPC6^T allele has been subject to artificial selection in modern East Asian japonica rice varieties. Collectively, these findings provide a crucial foundation for understanding the genetic mechanisms underlying the rice GPC variation and an important target gene for quality improvement practice.