The GS3 locus located in the pericentromeric region of rice chromosome 3 has been frequently identified as a major QTL for both grain weight (a yield trait) and grain length (a quality trait) in the literature. Near isogenic lines of GS3 were developed by successive crossing and backcrossing Minghui 63 (large grain) with Chuan 7 (small grain), using Minghui 63 as the recurrent parent. Analysis of a random subpopulation of 201 individuals from the BC3F2 progeny confirmed that the GS3 locus explained 80–90% of the variation for grain weight and length in this population. In addition, this locus was resolved as a minor QTL for grain width and thickness. Using 1,384 individuals with recessive phenotype (large grain) from a total of 5,740 BC3F2 plants and 11 molecular markers based on sequence information, GS3 was mapped to a DNA fragment approximately 7.9 kb in length. A full-length cDNA corresponding to the target region was identified, which provided complete sequence information for the GS3 candidate. This gene consists of five exons and encodes 232 amino acids with a putative PEBP-like domain, a transmembrane region, a putative TNFR/NGFR family cysteine-rich domain and a VWFC module. Comparative sequencing analysis identified a nonsense mutation, shared among all the large-grain varieties tested in comparison with the small grain varieties, in the second exon of the putative GS3 gene. This mutation causes a 178-aa truncation in the C-terminus of the predicted protein, suggesting that GS3 may function as a negative regulator for grain size. Cloning of such a gene provided the opportunity for fully characterizing the regulatory mechanism and related processes during grain development.