RNA-based therapeutics has attracted substantial interest from both academics and pharmaceutical companies. In this study, we investigated the function and the underlying mechanism of Gelsolin (GSN) 3'UTR in NSCLC H1299 and A549 cells. We found that transfected Flag-GSN plasmids significantly increased the proliferation, migration and invasion of NSCLC cells, whereas GSN 3'UTR could suppress the promotional effect of GSN protein on the development of NSCLC in vitro. Interestingly, we observed that these in vitro anticancer effects of GSN 3'UTR was independent of the co-expression with GSN coding sequence. Moreover, transfected GSN 3'UTR affected the actin-cytoskeleton remodeling and epithelial-mesenchymal transition (EMT) processes in H1299 and A549 cells, and targeted the co-expressed proteins to the plasma membrane. Subsequently, RNA pull-down assays have been performed to identify Tra2β protein as a GSN 3'UTR binder. We then showed that Tra2β was important for the localized protein expression mediated by GSN 3'UTR. Taken together, our results suggested that GSN 3'UTR may exert anticancer functions in NSCLC cells through regulating the subcellular localized expression of GSN protein mediated by the interaction between GSN 3'UTR-Tra2β.