Trimethylamine N-oxide (TMAO) is a well-known naturally occurring osmolyte in animals that counteracts the effect of different denaturants related to environmental stress and has recently been associated with severe human chronic diseases. In plants, however, the presence of TMAO has not yet been reported. In this study, we demonstrate that plants contain endogenous levels of TMAO, that it is synthesized by flavin-containing monooxygenases, and that its levels increase in response to abiotic stress conditions. In addition, our results reveal that TMAO operates as a protective osmolyte in plants, promoting appropriate protein folding and as an activator of abiotic stress-induced gene expression. Consistent with these functions, we show that TMAO enhances plant adaptation to low temperatures, drought, and high salt. We have thus uncovered a previously unidentified plant molecule that positively regulates abiotic stress tolerance.