Grain size is an important factor affecting yield production in rice. GS3， as the firstly cloned grain size gene in rice， mainly functions in regulation of the grain length and grain weight. GS3 protein contains OSR （Organ size regulation） domain at the N-terminal and Cys-rich domain at the C-terminal. There are four main natural variants of GS3， namely GS3-1 to GS3-4. GS3-1 encodes a full-length GS3 protein， GS3-2 encodes a protein with one amino acid missing， GS3-3 abolishes full-length GS3 protein （associating with longer seeds）， GS3-4 only encodes the N-terminal OSR domain （associating with significantly shorter seeds）. Up to now， the GS3 variant type encoding only the Cys-rich domain remains yet identified in the natural populations， and the consequence on seed size also remains unclear. In this study， by deployment of the CRISPR/Cas9 genome editing technology to delete large DNA fragments by two target sequences flanking the target DNA， we generated a new mutation variant gs3-5 that encodes only the C-terminal Cys-rich domain. The gs3-5 mutant showed increase on seed length and thousand grain weight， thus indicating that the OSR domain is necessary for the GS3 protein to act as a negative regulator of grain size. Collectively， these results provided a reference for further analysis of the regulation of the OSR structural domain on grain size and exploration of new breeding improvement methods.