As a major component of the plant primary cell wall, structure changes in pectin may affect the formation of the secondary cell wall and lead to serious consequences on plant growth and development. Pectin-modifying enzymes including pectate lyase-like proteins (PLLs) participate in the remodeling of pectin during organogenesis, especially during fruit ripening. In this study, we usedArabidopsisas a model system to identify criticalPLLgenes that are of particular importance for vascular development. FourPLLgenes, namedAtPLL15,AtPLL16,AtPLL19,andAtPLL26,were identified for xylem-specific expression. A knock-out T-DNA mutant ofAtPLL16displayed an increased amount of pectin, soluble sugar, and acid-soluble lignin (ASL). Interestingly, theatpll16mutant exhibited an irregular xylem phenotype, accompanied by disordered xylem ray cells and an absence of interfascicular phloem fibers. The xylem fiber cell walls in theatpll16mutant were thicker than those of the wild type. On the contrary,AtPLL16overexpression resulted in expansion of the phloem and a dramatic change in the xylem-to-phloem ratios. Altogether, our data suggest thatAtPLL16as a pectate lyase plays an important role during vascular development inArabidopsis.