Gastric cancer remains the second leading cause of cancer-related deaths worldwide. Although Helicobacter pylori (H. pylori) is considered to be a critical risk factor, the molecular mechanisms underlying H. pylori-induced gastric carcinogenesis are still poorly defined. Recently, accumulating studies have revealed that microRNAs play key roles in development, differentiation, immune regulation, and even carcinogenesis. This study was performed to explore the mechanism of microRNA-375 (miR-375) in H. pylori promotion of gastric carcinogenesis. It was shown that miR-375 was down-regulated in response to H. pylori infection in gastric epithelial cell lines; this finding was quite opposite to the expression patterns of pro-inflammatory cytokines observed in a co-culture cell model. Moreover, the ectopic expression of miR-375 aggravated cell proliferation and migration. It was further observed that Janus kinase 2 (JAK2) was a bona fide target of miR-375 and further activated signal transducer and activator of transcription 3 (STAT3) and other downstream target molecules. Both gain-of-function and loss-of-function experiments showed that decreased miR-375 expression could mimic the oncogenic effects of the JAK2-STAT3 pathway. In addition, pretreatment with siRNAs targeting JAK2 prevented gastric epithelial cells from increasing proliferation and migration even in response to H. pylori infection. For the first time, our results demonstrate that the JAK2-STAT3 pathway regulated by miR-375 is involved in H. pylori-induced inflammation; this pathway promotes neoplastic transformation by affecting the expression of BCL-2 and TWIST1, hence offering a potential therapeutic target for inflammation-related cancers, especially those related to H. pylori.