The rice cell wall is principally composed of cellulose, hemicellulose, lignin, and pectin, among which hemicellulose constitutes for approximately one-third of the total cell wall components. Xylan, the most abundant hemicellulosic polysaccharide, plays an important role in plant growth and development. In Arabidopsis, Glycosyltransferase 43 (GT43) and Glycosyltransferase 47 (GT47) families have been characterized as core components involved in the xylan backbone biosynthesis. To investigate the functions of the GT43 and GT47 family genes in rice xylan backbone synthesis, this study conducted comprehensive research on the family members OsIRX9L, OsIRX10, and OsIRX14 using bioinformatics, gene expression analysis, cell wall composition assays, and mutant phenotype characterization. The results revealed that OsIRX9L, OsIRX10, and OsIRX14 are highly evolutionarily conserved homologs of the Arabidopsis proteins AtIRX9L, AtIRX10, and AtIRX14, respectively. Gene expression analysis indicated that these three genes exhibit higher transcript levels in tissues rich in secondary wall and showed a high co-expression pattern with CESAs required for secondary wall cellulose biosynthesis. Using CRISPR/Cas9 gene-editing technology, homozygous mutant lines (osirx9l-1, osirx9l-2, osirx10-1, osirx10-2, osirx14-1, and osirx14-2) for these genes were generated. Immunohistochemical staining assay showed that, compared to the wild-type, all mutants showed significantly reduced xylan content, as well as a marked decrease in xylose levels. Among them, the osirx10 mutants exhibited the lowest xylan and xylose contents. Cell wall composition analysis further indicated that both hemicellulose and cellulose contents were lower in the mutants than in wild-type, with a particularly significant reduction in hemicellulose. The osirx10 mutants showed the most significant decrease. Agronomic trait measurements revealed that the mutants exhibited a significant reduction in plant height and dry weight compared to wild-type. In summary, this study demonstrates that the OsIRX9L, OsIRX10, and OsIRX14 genes collectively contribute to the biosynthesis of xylan in rice, significantly influencing cell wall composition and plant growth and development. Among them, OsIRX10 plays a predominant role.