Urease is involved in Helicobacter pylori infection and survival in acid circumference. This study explored the overexpression of H. pylori-associated urease mRNAs in human gastric cancers by using a well-established membrane array analysis method in our lab. Analysis of 30 gastric cancer tissue specimens and 30 paired adjacent normal tissues demonstrated that urease genes involved in H. pylori infection were upregulated in gastric cancer tissues. UreA, G, and I are predominant genotypes found in gastric cancer tissues. However, the mRNA levels of UreC and UreE were hardly to be found in both gastric cancer and normal tissues in our study. In addition, we treated NIH-3T3 cells with two kinds of H. pylori exudates [weak urease activity (HP-W) and strong urease activity (HP-S)], which contained 1.6, 3.1, 6.5, 13, and 25.9 pg/mL urease of HP-W exudates and 18, 36, 75, 150, and 300 pg/mL urease of HP-S exudates. NIH-3T3 cells were treated with these different concentration components for 24 h. Cell proliferation rate was elevated 2.7%, 9.9%, 18.9%, 36.6%, and 42.9%, respectively, after HP-W exudates were treated, and elevated 8.1%, 31.9%, 45.9%, 74.9%, and 81.3%, respectively, after treatment with HP-S exudates. In further investigation of the time course of NIH-3T3 cells treated with 50 microg/mL H. pylori, the exudates revealed that the proliferation rate was elevated 14%, 23.7%, 38.7%, 31.6%, and 29%, respectively, after HP-W treatment and elevated 29.8%, 50.4%, 78.5%, 62.3%, and 55.9% after HP-S treatment for 6, 12, 24, 48, and 72 h, respectively. In conclusion, membrane array promises a new diagnostic tool to detect H. pylori more sensitively than the CLO test. These results suggest that urease may play an important role in the development of gastric mucosal hyperproliferation in H. pylori-induced gastritis.