<p>Endosperm development in rice is under the control of a complex network of genes that orchestrate starch biosynthesis and determine grain quality. In this study, we identified a gamma-ray induced rice mutant, <i>ARTI-flo1</i>, characterized by a striking floury-white endosperm phenotype. Genetic analysis using MutMap identified a C-to-T transition in the intron of cytosolic pyruvate orthophosphate dikinase (<i>OsPPDKB</i>), a key gene regulating carbon allocation and starch synthesis in the rice endosperm. Structural and biochemical assays revealed that <i>ARTI-flo1</i> grains possess loosely packed starch granules and show marked reductions in amylose and damaged starch contents, indicating disrupted starch architecture. To validate the causative role of <i>ARTI-flo1</i>, we generated CRISPR-Cas9-mediated knockout lines, which recapitulated the floury phenotype. Transcriptomic profiling demonstrated that the cytosolic isoform of OsPPDKB plays a pivotal role in starch biosynthesis, as the expression of genes involved in both amylose and amylopectin synthesis was substantially diminished in <i>ARTI-flo1</i> grains. Comparative haplotype analysis across 3,000 cultivated rice accessions identified two major <i>ARTI-flo1</i> haplotypes. However, neither was significantly associated with the floury phenotype, suggesting that environmental factors may play a stronger role than natural allelic variation. Collectively, our findings establish <i>ARTI-flo1</i> as a key regulator of endosperm structure and starch composition in rice. Moreover, the successful reconstruction of this allele via genome editing underscores the utility of CRISPR-based approaches for functional gene validation and for creating new allelic variants to enhance grain quality traits.</p>