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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, <i>qTGW10-20.8</i>, was mapped in a 70.7 kb region on chromosome 10. Validation of the candidate gene for <i>qTGW10-20.8</i>, <i>OsMADS56</i> encoding a MADS-box transcription factor, was performed in this study. In a near-isogenic line (NIL) population segregated only at the <i>OsMADS56</i> locus, NILs carrying the <i>OsMADS56</i> allele of IRBB52 were 1.9% and 2.9% lower in TGW than NILs carrying the <i>OsMADS56</i> allele of Teqing in 2018 and 2020, respectively. Using <i>OsMADS56</i> knock-out mutants and overexpression transgenic plants, <i>OsMADS56</i> was validated as the causal gene for <i>qTGW10-20.8</i>. 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 <i>OsMADS56</i>. In transgenic populations of <i>OsMADS56</i> 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 <i>OsMADS56</i> is the major type presented in cultivated rice and used in variety improvement. Cloning of <i>OsMADS56</i> provides a new gene resource to improve grain weight and size through molecular design breeding.