Gastrokine-2 (GKN2) is a secretory protein that is decreased or absent in gastric cancer tissues. In addition, it is reported that trefoil factor 1 (TFF1) and TFF2 can both bind GKN2. In this study, we investigated the expression and biological functions of GKN2 and its interaction with TFF2 in gastric cancer. We found that GKN2 expression was significantly downregulated or absent in gastric cancer cell lines, gastric intestinal metaplasia, and tumor tissues. Overexpression of GKN2 suppressed the proliferation, migration, and invasion of gastric cancer cells and arrested the cell cycle at the G1-S transition phase. Furthermore, GKN2 efficiently attenuated tumor growth in SGC-7901 nude mice xenograft models. Overexpression of GKN2 also reduced the expression levels of cylinD1, cyclinE1, and matrix metalloproteinase 9 (MMP9), which were correlated with proliferation and metastasis of cancer cells. In co-transfected cells, TFF2 and GKN2 did not bind to each other. Overexpression of both GKN2 and TFF2 showed the same inhibitory effect as overexpression of GKN2 alone. Taken together, these findings suggested that GKN2 could inhibit the proliferation, migration, and invasion of gastric cancer cells and might represent a novel therapeutic target for gastric cancer. TFF2 may not interact or cooperate with GKN2, either structurally or functionally.