Intestinal trefoil factor (TFF3) is essential in regulating cell migration and maintaining mucosal integrity in gastrointestinal tract. We previously showed that TFF3 was overexpressed in gastric carcinoma. Whether TFF3 possesses malignant potential is not fully elucidated. We sought to investigate the effects of inducting TFF3 expression in a non-malignant rat fibroblast cell line (Rat-2) on the cell proliferation, invasion and the genes regulating cell invasion. Invasiveness and proliferation of transfected Rat-2 cell line were assessed using in vitro invasion chamber assay and colorimetric MTS assay. Differential mRNA expressions of invasion-related genes, namely, metalloproteinases (MMP-9), tissue inhibitors of metalloproteinases (TIMP-1), beta-catenin and E-cadherin, were determined by quantitative real-time polymerase chain reaction (PCR). We showed that TFF3 did not inhibit the proliferation of Rat-2 cells. We also demonstrated that transfection of TFF3 significantly promoted invasion of Rat-2 cells by 1.4- to 2.2-folds. There was an upregulation of beta-catenin (13.1-23.0%) and MMP-9 (43.4-92.2%) mRNA expression levels, and downregulation of E-cadherin (25.6-33.8%) and TIMP-1 (31.5-37.8%) in TFF3-transfected cells compared to controls during 48-h incubation. Our results suggested that TFF3 possesses malignant potential through promotion of cell invasiveness and alteration of invasion-related genes.