Abstract text: Glucuronic acid substitution of xylan is an important factor influencing cell wall recalcitrance. In Leymus chinensis (sheepgrass), this modification progressively increases during development and is accompanied by a decline in digestibility. Comparative analysis with Arabidopsis and rice GUX (GlucUronic acid substitution of Xylan) proteins revealed a significant expansion of the GUX gene family. Based on phylogenetic relationships and expression profiles, three pairs of highly expressed genes were designated LcGUX1–3. LcGUX1 is predominantly expressed in young stems at the booting stage, whereas LcGUX2 is mainly expressed in mature stems at heading, indicating functional divergence. To elucidate their roles, LcGUX1/2 were heterologously expressed in rice under the constitutive promoter pACT1 and the vascular-specific promoter pBS1 and Arabinoglytransferase promoter pXAT4. PACE result revealed increased glucuronic acid substitution of xylan and reduced glucose release, with distinct positional preferences. CRISPR/Cas9 was used to generated Lcgux mutants. Lcgux1 reduced substitution by 20–25%, whereas Lcgux2 decreased substitution by 60–70% and nearly abolished substituted xylo-oligosaccharides, significantly enhancing glucose release without affecting xylose content. These findings identify xylan structure as a critical target for improving digestibility and provide valuable genetic resources for forage crop breeding.