To investigate the biological activity of the H. pylori SlyD in vitro.Helicobacter pylori (H.pylori) slyD prokaryotic expression vector was carried out in Escherichia coli (E.coli), and recombination SlyD (rSlyD) was purified by immobilized metal affinity chromatography. The proliferation, apoptosis, invasion, transformation effects of rSlyD on AGS cells was detected by CCK-8, cell cycle, caspase-3 activity, matrigel invasion assay, and double-deck soft agar colony forming efficiency. In addition, the expressions of PCNA, KI-67, caspase-3, and MMP-9 were detected by western blot and immunofluorescence assay, respectively.The CCK-8 assay revealed that cell proliferation was increased in a time and dose-dependent manner in AGS + rSlyD group compared with that of AGS or AGS + PBS group (p.05). There are significant difference of PCNA and KI67 expressions among AGS, AGS + PBS, AGS + rSlyD groups (p.05). Soft agar colony formation assay revealed the colony number (foci100 μm) in AGS + rSlyD group was 26.3 ± 7.09, whereas 5.6 ± 1.15 in AGS and 5.0 ± 1.0 in AGS + PBS groups, respectively (p.01). Colorimetric enzyme assay revealed the activity of caspase-3 was decreased to 31.45 ± 0.49 after treatment with rSlyD, whereas 55.5 ± 0.43 in AGS and 55.1 ± 0.25 in AGS + PBS group, respectively (p.001). Similar caspase-3 expression also was confirmed by Western blot. The number of invasive cells in transwell chambers assay is 196.66 ± 40.41 in AGS + rSlyD group higher than 85 ± 22.9 in AGS or 81.66 ± 15.27 in AGS + PBS group, respectively (p.001). The MMP-9 expression in AGS + rSlyD group was also higher than that of AGS or AGS + PBS group.These results suggest that the HpSlyD may play an important role in disturbing cell proliferation, apoptosis, and enhancing cell transformation and invasion in the AGS cell line. HpSlyD might contribute to gastric pathogenicity in H.pylori-associated diseases.