Virulence associated protein D has been studied in the chromosome or plasmid of many species, and two different putative virulence associated protein D, HP0315 and HP0967, in Helicobacter pylori 26695 were reported in TIGR. However, the relationship and bio-functions between HP0315 and HP0967 remained largely unknown elements, and there were no published data to support their functions. Therefore, it was worth to investigate the functions and study the interaction between each other or other virulence factors. To study their functions, recombinant HP0315 and HP0967 were constructed, purified and assayed. The deduced amino acid sequence of either HP0315 or HP0967 shares less than 35.5% sequence identity with that of virulence associated protein D from several other species, for example Neisseria meningitidis MC58 and Haemophilus influenzae. About 49.5% identity was found in deduced amino acid sequence between HP0315 and HP0967. The HP0315 and HP0967 open reading frame (285 bp and 288bp) were individually cloned into the pQE30 vector and overexpressed in Escherichia coli strain SG13009. The results of N-terminally 6xHis-tagged HP0315 protein (12.58 kDa) and HP0967 (12.17 kDa) were purified by Ni–NTA affinity chromatography at a yield of 10 mg/L of bacteria culture, respectively. Analytical ultracentrifugation has shown that the HP0315 protein exists as a monomer in solution pH 3 and HP0967 appeared as a monomer, too. The HP0315 is easy to precipitate below its pI (at pH 6.7), and the HP0967 is more stable at this pH. Both recombinant HP0315 and HP0967 protein dominated with alpha-helix structure (35.3% and 52.7%) at pH 8, respectively. At pH 3, more apparent change in secondary structure was observed in Circular Dichroism spectra for HP0315 than for HP0967. Both recombinant HP0315 and HP0967 protein dominated with alpha-helix structure (66.3% and 50.7%) at pH 3, respectively. About 20 and 60 % cytotoxicity (corresponding to 80 and 40% survival fraction, respectively) were observed in Adenocarcinoma (AGS) cells after 48 hr co-culture with 150 and 300 μg/ml HP0315 protein (30 min acid-activation and neutralization previously) for 48 hr. Recombinant HP0315 (rec-HP0315) and recombinant HP0967 (rec-HP0967) proteins in AGS cells after co-culture were detected by Flow cytometry or immuno-fluorescence staining with antibodies against rec-HP0315 and rec-HP0967, respectively. Cross reaction to antibodies against (protocol a) HP0315 or (protocol b) HP0967 was also observed when AGS cells exposed to (protocol a) HP0967 or (protocol b) HP0315 for 24-48 hr. The mechanisms to affect cell proliferation of the virulence associated protein HP0315 remained to be further investigated. HP0315 protein could be detected in various stages H. pylori bacteria by means of western analysis. More HP0315 protein in the 24 hr spiral form than 48 hr coccoid form from suspension at pH 7.2 (ratio = 5:1) was observed. Acid-inducible phenomenon (10-fold) was observed in bacteria cultured on agar plates for 48 h at initial pH 5.5, compared to those at initial pH 7.2. In addition, acid-inducible HP0315 mRNA or HP0967 was identified also from log stage H. pylori after 2 hr culture on brucella agar plates at pH 5.5.