This dataset contains mass spectra (.raw) and mass spectrometry imaging (MSI) data (.imzML) from a deuterium isotope labeling experiment. The ‘.imzML files’ are used to generate MS images using MSiReader. The data files are split across four folders, along with a separate .xlsx analysis file, which correspond to the accompanying article, "Mass spectrometry imaging of Arabidopsis thaliana with in vivo D2O labeling." This study demonstrates that MSIi can reveal spatiotemporal metabolic activities in various tissues of the A. thaliana plant.