Xyloglucan endotransglucosylases/hydrolases (XTHs) are a class of enzymes capable of catalyzing the molecular grafting between xyloglucans and/or the endotype hydrolysis of a xyloglucan molecule. They are encoded by 33 genes in Arabidopsis. Whereas recent studies have revealed temporally and spatially specific expression profiles for individual members of this family in plants, their biological roles are still to be clarified. To identify the role of each member of this gene family, we examined phenotypes of mutants in which each of the Arabidopsis XTH genes was disrupted. This was undertaken using a reverse genetic approach, and disclosed two loss-of-function mutants for the AtXTH27 gene, xth27-1 and xth27-2. These exhibited short-shaped tracheary elements in tertiary veins, and reduced the number of tertiary veins in the first leaf. In mature rosette leaves of the mutant, yellow lesion-mimic spots were also observed. Upon genetic complementation by introducing the wild-type XTH27 gene into xth27-1 mutant plants, the number of tertiary veins was restored, and the lesions disappeared completely. Extensive expression of the pXTH27::GUS fusion gene was observed in immature tracheary elements in the rosette leaves. The highest level of AtXTH27 mRNA expression in the rosette leaves was observed during leaf expansion, when the tracheary elements were elongating. These findings indicate that AtXTH27 plays an essential role during the generation of tracheary elements in the rosette leaves of Arabidopsis.