Endometrial cancer is the most common gynecologic malignancy in women worldwide. In the present study, we evaluated the effects of neural stem cell-directed enzyme/prodrug therapy (NDEPT) designed to more selectively target endometrial cancer. For this, we employed two different types of neural stem cells (NSCs), HB1.F3.CD and HB1.F3.CD.IFN-β cells. Cytosine deaminase (CD) can convert the non-toxic prodrug, 5-fluorocytosine (5-FC), into a toxic agent, 5-fluorouracil (5-FU), which inhibits DNA synthesis. IFN-β is a powerful cytotoxic cytokine that is released by activated immune cells or lymphocytes. In an animal model xenografted with endometrial Ishikawa cancer cells, the stem cells stained with CM-DiI were injected into nearby tumor masses and 5-FC was delivered by intraperitoneal injection. Co-expression of CD and IFN-β significantly inhibited the growth of cancer (~50–60%) in the presence of 5-FC. Among migration-induced factors, VEGF gene was highly expressed in endometrial cancer cells. Histological analysis showed that the aggressive nature of cancer was inhibited by 5-FC in the mice treated with the therapeutic stem cells. Furthermore, PCNA expression was more decreased in HB1.F3.CD.IFN-β treated mice rather than HB1.F3.CD treated mice. To confirm the in vitro combined effects of 5-FU and IFN-β, 5-FU was treated in Ishikawa cells. 5-FU increased the IFN-β/receptor 2 (IFNAR2) and BXA levels, indicating that 5-FU increased sensitivity of endometrial cancer cells to IFN-β, leading to apoptosis of cancer cells. Taken together, these results provide evidence for the efficacy of therapeutic stem cell-based immune therapy involving the targeted expression of CD and IFN-β genes at endometrial cancer sites.