Grain weight and size are important traits determining grain yield and influencing grain quality in rice. In a previous study, a quantitative trait locus controlling thousand-grain weight (TGW) in rice, qTGW10-20.8, was mapped in a 70.7 kb region on chromosome 10. Validation of the candidate gene for qTGW10-20.8, OsMADS56 encoding a MADS-box transcription factor, was performed in this study. In a near-isogenic line (NIL) population segregated only at the OsMADS56 locus, NILs carrying the OsMADS56 allele of IRBB52 were 1.9% and 2.9% lower in TGW than NILs carrying the OsMADS56 allele of Teqing in 2018 and 2020, respectively. Using OsMADS56 knock-out mutants and overexpression transgenic plants, OsMADS56 was validated as the causal gene for qTGW10-20.8. Compared with the recipients, the TGW of the knock-out mutants was reduced by 6.0–15.0%. In these populations, decreased grain weight and size were associated with a reduction in the expression of OsMADS56. In transgenic populations of OsMADS56 driven by a strong constitutive promoter, grain weight and size of the positive plants were significantly higher than those of the negative plants. Haplotype analysis showed that the Teqing-type allele of OsMADS56 is the major type presented in cultivated rice and used in variety improvement. Cloning of OsMADS56 provides a new gene resource to improve grain weight and size through molecular design breeding.