The ebb-and-flow subirrigation (EFI) is a water-saving and environment-friendly irrigation method, which can effectively improve water use efficiency and promote plant growth. In this study, we elucidated the effects of ebb-and-flow subirrigation on the protein levels in tomato roots in comparison with top sprinkle irrigation (TSI), and used an integrated approach involving tandem mass tag (TMT) labeling, high performance liquid chromatography (HPLC) fractionation, and mass spectrometry (MS) based analysis. A total of 8,510 quantifiable proteins and 513 dif-ferentially accumulated proteins (DAPs) were identified, of which the expressions of 283 DAPs were up-regulated, and 230 DAPs were down-regulated in the EFI vs TSI treatment comparison. According to proteomic data, we performed a systematic bioinformatics analysis of all identified proteins and DAPs. The DAPs were most significantly associated with the terms “metabolic process”, “anchored component of membrane”, “oxidoreductase activity”, “phenylpropanoid biosynthesis” and “biosynthesis of secondary metabolites” according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes enrichment (KEGG) analysis. The 272 DAPs were classified into 12 subcellular components according to their subcellular localizations.