Grain size and weight are critical determinants of rice yield and quality, yet their underlying genetic and regulatory mechanisms remain largely unexplored. In this study, we identified a protein containing TPR domain, named OsTPR028, which is localized in the cytoplasm. The gene encoding this protein is highly expressed during grain development. Homozygous ostpr028 mutant exhibited significant reductions in grain size, grain filling rate, and grain weight, accompanied by decreased levels of starch, amylose, and lipids. Transcriptomic and qRT-PCR analyses demonstrated that OsTPR028 regulates the expression of genes involved in starch biosynthesis and lipid transport. Protein interaction assays revealed that OsTPR028 interacts with an endosperm-specific protein, OsEnS45, whose knockout similarly resulted in reduced grain size and weight. Further investigations indicated that impaired spikelet cell division is the primary cause of these phenotypic defects in both ostpr028 and osens45 mutants. Together, our findings elucidate the critical role of the OsTPR028-OsEnS45 module in grain development and offer promising molecular targets for improving rice yield and quality through breeding programs.