Mutation of OsANK3 increases grain length, grain weight, and chalkiness. OsANK3 influences grain size and quality by regulating genes involved in the cell cycle and starch metabolism. Grain size and endosperm starch content are key determinants of rice yield and quality. In this study, we investigated the function of OsANK3, a gene encoding ankyrin repeats, in regulating grain development traits. This gene was initially identified through mass spectrometry analysis as a potential upstream regulator of rice chalkiness in our previous screening. Using CRISPR/Cas9 technology, we generated OsANK3 knockout mutants (cr-osank3-2, cr-osank3-6, and cr-osank3-7) and found that OsANK3 is predominantly expressed in stems and leaves, with subcellular localization in the cytoplasm and plasma membrane. Disruption of OsANK3 increased plant height, grain length, grain weight, and chalkiness while reducing total starch content, amylose content (AC), and gel consistency (GC). Cytological analysis revealed that the elongated grains in cr-osank3 mutants resulted from enhanced cell proliferation and elongation in the outer lemma. qRT-PCR data demonstrated that OsANK3 regulates cell cycle-related genes, thereby influencing cell division and expansion. In addition, starch biosynthesis genes (OsGBSSI, OsAGPL1) were down-regulated in mutants, whereas starch hydrolase genes (OsAmy1 A, OsAmy3B) were up-regulated. Our findings demonstrate that OsANK3 knockout enhances grain size but compromises grain quality by altering cell dynamics and starch metabolism. This study elucidates the molecular role of OsANK3 in grain development and provides a valuable target for rice breeding programs.