Interest in gene therapy has recently increased; in particular, gene-directed enzyme/prodrug therapies have been found to be more advantageous compared to radiotherapy and/or chemotherapy. One of these, a cytosine deaminase (CD)/5-fluorocytosine (5-FC) system, is known to induce apoptosis by converting 5-FC, a prodrug, to its metabolically active form, 5-fluorouracil. The present study was designed to examine the migratory and therapeutic effects of engineered human stem cell lines against endometrial cancer using this strategy. The parental stem cells, HB1.F3, were modified to express Escherichia coli CD or human interferon-beta (IFN-β), thereby producing HB1.F3.CD and HB1.F3.CD.IFN-β cells, respectively. The parental and engineered stem cells (HB1.F3, HB1.F3.CD, and HB1.F3.CD.IFN-β) significantly migrated toward endometrial cancer cells (Ishikawa) more than primary bovine fibroblasts (bovine FB). In addition, important chemoattractant factors, including stem cell factor (SCF), vescular endothelial growth factor, vescular endothelial growth factor receptor 2, C-X-C chemokine receptor type 4, and c-KIT, involved in the tumor-tropic ability of stem cells were expressed in Ishikawa cells. In using a co-culture system and MTT assay, reduced viability of endometrial cancer cells was observed in the presence of HB1.F3.CD and HB1.F3.CD.IFN-β cells with prodrug 5-FC. Taken together, these results suggest that gene therapy employing genetically modified stem cells expressing CD and IFN-β may be effective for treating endometrial cancer.