Trehalose exists in most living organisms and functions as a storage carbohydrate and as an osmoprotectant in yeast, fungi, and bacteria. Trace amount of endogenous trehalose was detected in flowering plants, and the trehalose biosynthetic pathway was essential for embryo maturation in Arabidopsis. Conversely, exogenous trehalose was toxic to higher plants and severely curtailed root and shoot growth. In the current study, 30 mM trehalose was added to 2-week-old liquid cultures containing Arabidopsis thaliana (Columbia ecotype) seedlings. Densely stained granular particles were detected in the extracellular space of cotyledons and roots of trehalose-treated seedlings using transmission electron microscopy. Expression levels of 91 transcripts were altered by 1–6 h of trehalose treatment using DNA microarray analysis, and 65 of these encoded either known proteins or putative proteins with known functions. The exogenous trehalose treatment altered transcript levels of transcription factors, cell wall modification, nitrogen metabolism, and stress-related, defense-related, and fatty acid biosynthesis genes. Many of the transcripts altered by exogenous trehalose treatment were associated with the ethylene and methyl jasmonate-signaling pathways. The above findings suggested that trehalose, or metabolites derived from trehalose, are important regulators of plant gene expression in higher plants.