Grain protein content (GPC) affects rice nutrition quality. Here, we identify two stable quantitative trait loci (QTLs),
qGPC-1
and
qGPC-10
, controlling GPC in a mapping population derived from
indica
and
japonica
cultivars crossing. Map-based cloning reveals that
OsGluA2
, encoding a glutelin type-A2 precursor, is the candidate gene underlying
qGPC-10
. It functions as a positive regulator of GPC and has a pleiotropic effect on rice grain quality. One SNP located in
OsGluA2
promoter region is associated with its transcript expression level and GPC diversity. Polymorphisms of this nucleotide can divide all haplotypes into low (
OsGluA2
LET
) and high (
OsGluA2
HET
) expression types. Population genetic and evolutionary analyses reveal that
OsGluA2
LET
, mainly present in
japonica
accessions, originates from wild rice. However,
OsGluA2
HET
, the dominant type in
indica
, is acquired through mutation of
OsGluA2
LET
. Our results shed light on the understanding of natural variations of GPC between
indica
and
japonica
subspecies.