Among 1590 ORFs in the Helicobacter pylori genome, >250 have been identified as authentic genes by proteomic analysis. Low-abundance proteins need to be enriched to a minimal amount for MALDI-TOF analysis and salt precipitation has generally been used for protein enrichment. Here, a whole-cell extract of H. pylori strain 26695 was subjected to protein fractionation with stepwise concentrations of ammonium sulfate and the proteins were displayed by 2-DE. The protein spots were quantified using PDQUEST software and identified by peptide fingerprinting. The 2-DE profiles and intensities of individual protein spots differed among the protein fractions. Out of the 98 identified proteins, 61 were found in the stepwise ammonium sulfate fractions but not in the whole-cell extract. Out of these, 37 proteins, including KdsA, were found exclusively in a single fraction. In contrast, GroEL, UreA, UreB, TrxA, NapA, and FldA were ubiquitously present in all fractions. Iron-containing proteins such as NapA, SodB, CeuE, and Pfr were found predominantly in the 100% saturated ammonium sulfate precipitate. Additionally, 29 proteins were newly identified in this study. These data will facilitate the preparation of significant H. pylori proteins, as well as provide information about low-abundance proteins.