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Cellulose is an abundant component of plant cell wall matrices, and this para-crystalline polysaccharide is synthesized at the plasma membrane by motile Cellulose Synthase Complexes (CSCs). However, the factors that control CSC activity and motility are not fully resolved. In a targeted chemical screen, we identified the alkylated nojirimycin analog N -Dodecyl Deoxynojirimycin (ND-DNJ) as a small molecule that severely impacts Arabidopsis seedling growth. Previous work suggests that ND-DNJ-related compounds inhibit the biosynthesis of glucosylceramides (GlcCers), a class of glycosphingolipid associated with plant membranes. Our work uncovered major changes in the sphingolipidome of plants treated with ND-DNJ, including reductions in GlcCer abundance and altered acyl chain length distributions. Crystalline cellulose content was also reduced in ND-DNJ-treated plants as well as plants treated with the known GlcCer biosynthesis inhibitor N-[2-hydroxy-1-(4-morpholinylmethyl)-2-phenyl ethyl]-decanamide (PDMP) or plants containing a genetic disruption in GLUCOSYLCERAMIDE SYNTHASE (GCS), the enzyme responsible for sphingolipid glucosylation that results in GlcCer synthesis. Live-cell imaging revealed that CSC speed distributions were reduced upon treatment with ND-DNJ or PDMP, further suggesting an important relationship between glycosylated sphingolipid composition and CSC motility across the plasma membrane. These results indicate that multiple interventions compromising GlcCer biosynthesis disrupt cellulose deposition and CSC motility, suggesting that GlcCers regulate cellulose biosynthesis in plants. [ABSTRACT FROM AUTHOR]