The vascular system of plants consists of two conducting tissues, xylem and phloem, which differentiate from procambium cells. Xylem serves as a transporting system for water and signaling molecules and is formed by sequential developmental processes, including cell division/expansion, secondary cell wall deposition, vacuole collapse, and programmed cell death (PCD). PCD during xylem differentiation is accomplished by degradation of cytoplasmic constituents, and it is required for the formation of hollow vessels, known as tracheary elements (TEs). Our recent study revealed that the small GTPase RabG3b acts as a regulator of TE differentiation through its autophagic activation. By using an Arabidopsis in vitro cell culture system, we showed that autophagy is activated during TE differentiation. Overexpression of a constitutively active RabG3b (RabG3bCA) significantly enhances both autophagy and TE differentiation, which are consistently suppressed in transgenic plants overexpressing a dominant negative form (RabG3bDN) or RabG3bRNAi (RabG3bRNAi), a brassinosteroidinsensitive mutant bri1-301, and an autophagy mutant atg5-1. Wood (called secondary xylem) is the most abundant biomass produced by land plants including Populus and Eucalyptus, and therefore is considered to be one of the most cost-effective and renewable bioenergy resources. In an attempt to enhance xylem differentiation and thus to improve biomass traits in poplars, we generated transgenic poplars overexpressing the RabG3bCA form. As notable phenotypes, both stem height and diameter were increased and xylem area in vascular bundles was significantly expanded in RabG3bCA transgenic poplars compared to control plants. Taken together, these results demonstrate that RabG3b regulates xylem differentiation in both Arabidopsis and Populus. This study enhances our understanding of biological mechanisms underlying wood formation and serve as a framework to engineer the quality and quantity of wood as useful biomass.