Emerging evidence has shown the association of aberrantly expressed microRNAs (miRNAs) with tumor development and progression. However, little is known about the potential role of miRNAs in gastric carcinogenesis. Here, we performed miRNA microarray to screen miRNAs differentially expressed in the paired gastric cancer and their adjacent nontumor tissues and found that miR-375 was greatly downregulated in gastric cancer tissues. Quantitative real-time PCR analysis verified that miR-375 expression was significantly decreased in more than 90% of primary gastric cancers compared with their nontumor counterparts from patients undergoing gastric resection. Overexpression of miR-375 significantly inhibited gastric cancer cell proliferation in vitro and in vivo. Forced expression of miR-375 in gastric cancer cells significantly reduced the protein level of Janus kinase 2 (JAK2) and repressed the activity of a luciferase reporter carrying the 3'-untranslated region of JAK2, which was abolished by mutation of the predicted miR-375-binding site, indicating that JAK2 may be a miR-375 target gene. Either inhibition of JAK2 activity by AG490 or silencing of JAK2 by RNAi suppressed gastric cancer cell proliferation resembling that of miR-375 overexpression. Moreover, ectopic expression of JAK2 can partially reverse the inhibition of cell proliferation caused by miR-375. Finally, we found a significant inverse correlation between miR-375 expression and JAK2 protein level in gastric cancer. Thus, these data suggest that miR-375 may function as a tumor suppressor to regulate gastric cancer cell proliferation potentially by targeting the JAK2 oncogene, implicating a role of miR-375 in the pathogenesis of gastric cancer.