Efforts to increase glycinebetaine (GB) levels in plants have been pursued as an approach to improving plant performance under stress conditions. To date, the impact of engineered levels of GB has been limited by metabolic constraints that restrict the achieved increases. We report the identification of a novel gene, GB1, that is differentially expressed in high and low GB accumulating maize genotypes. The predicted GB1 protein shows 60% identity to a putative C-4 sterol methyl oxidase from rice. Overexpression of GB1 in maize and soybean led to dramatically higher leaf GB content in most of the transgenic lines compared to wild-type. These results suggest that the GB1 protein is an important component of the biochemical pathways controlling GB accumulation in plants.