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Cellulose synthesis is a complex process in plant cells that is important for wood processing, pulping, and papermaking. Cellulose synthesis begins with the glycosylation of sitosterol by sitosterol glycosyltransferase (SGT) to produce sitosterol-glucoside (SG), which acts as the guiding primer for cellulose production. However, the biological functions of SGTs in <i>Populus tomentosa</i><i>(P. tomentosa)</i> remain largely unknown. Two full-length <i>PtSGT</i> genes (<i>PtSGT1</i> and <i>PtSGT4</i>) were previously isolated from <i>P. tomentosa</i> and characterized. In the present study, CRISPR/Cas9 gene-editing technology was used to construct <i>PtSGT1-</i>sgRNA and <i>PtSGT4-</i>sgRNA expression vectors, which were genetically transformed into <i>P. tomentosa</i> using the <i>Agrobacterium</i>-mediated method to obtain transgenic lines. Nucleic acid and amino acid sequencing analysis revealed both base insertions and deletions, in addition to reading frame shifts and early termination of translation in the transgenic lines. Sugar metabolism analysis indicated that sucrose and fructose were significantly downregulated in stems and leaves of mutant <i>PtSGT1-</i>1 and <i>PtSGT4-</i>1. Glucose levels did not change significantly in roots and stems of <i>PtSGT1-</i>1 mutants; however, glucose was significantly upregulated in stems and downregulated in leaves of the <i>PtSGT4-</i>1 mutants. Dissection of the plants revealed disordered and loosely arranged xylem cells in the <i>PtSGT4-</i>1 mutant, which were larger and thinner than those of the wild-type. This work will enhance our understanding of cellulose synthesis in the cell walls of woody plants.