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The endosperm is the main nutrient source in cereals for humans, as it is a highly specialized storage organ for starch, lipids, and proteins, and plays an essential role in seed growth and development. Active DNA demethylation regulates plant developmental processes and is ensured by cytosine methylation (5-meC) DNA glycosylase enzymes. To find out the role of <i>OsROS1a</i> in seed development, the null mutant of <i>OsROS1a</i> was generated using the CRISPR/Cas9 system. The null mutant of <i>OsROS1a</i> was stable and heritable, which affects the major agronomic traits, particularly in rice seeds. The null mutant of <i>OsROS1a</i> showed longer and narrower grains, and seeds were deformed containing an underdeveloped and less-starch-producing endosperm with slightly irregularly shaped embryos. In contrast to the transparent grains of the wild type, the grains of the null mutant of <i>OsROS1a</i> were slightly opaque and rounded starch granules, with uneven shapes, sizes, and surfaces. A total of 723 differential expression genes (DEGs) were detected in the null mutant of <i>OsROS1a</i> by RNA-Seq, of which 290 were downregulated and 433 were upregulated. The gene ontology (GO) terms with the top 20 enrichment factors were visualized for cellular components, biological processes, and molecular functions. The key genes that are enriched for these GO terms include starch synthesis genes (<i>OsSSIIa</i> and <i>OsSSIIIa</i>) and cellulose synthesis genes (<i>CESA2</i>, <i>CESA3</i>, <i>CESA6</i>, and <i>CESA8</i>). Genes encoding polysaccharides and glutelin were found to be downregulated in the mutant endosperm. The glutelins were further verified by SDS-PAGE, suggesting that glutelin genes could be involved in the null mutant of <i>OsROS1a</i> seed phenotype and <i>OsROS1a</i> could have the key role in the regulation of glutelins. Furthermore, 378 differentially alternative splicing (AS) genes were identified in the null mutant of <i>OsROS1a</i>, suggesting that the <i>OsROS1a</i> gene has an impact on AS events. Our findings indicated that the function on rice endosperm development in the null mutant of <i>OsROS1a</i> could be influenced through regulating gene expression and AS, which could provide the base to properly understand the molecular mechanism related to the <i>OsROS1a</i> gene in the regulation of rice seed development.