This study was conducted to investigate enhancement of anti-tumor effects of the lentogenic Newcastle disease virus Clone30 strain (NDV rClone30) expressing cytosine deaminase (CD) gene against tumor cells and in murine groin tumor-bearing models. Cytotoxic effects of the rClone30-CD/5-FC on the HepG2 cell line were examined by an MTT method. Anti-tumor activity of rClone30-CD/5-FC was examined in H22 tumor-bearing mice. Compared to the rClone30-CD virus treatment alone, NDV rClone30-CD/5-FC at 0.1 and 1 MOIs exerted significant cytotoxic effects (P<0.05) on HepG2 cells. For treatment of H22 tumor-bearing mice, recombinant NDV was injected together with 5-FC given by either intra-tumor injection or tail vein injection. When 5-FC was administered by intra-tumor injection, survival for the rClone30-CD/5-FC-treated mice was 4/6 for 80 days period vs 1/6, 0/6 and 0/6 for the mice treated with rClone30-CD, 5-FC and saline alone, respectively. When 5-FC was given by tail vein injection, survival for the rClone30-CD/5-FC-treated mice was 3/6 vs 2/6, 0/6 and 0/6 for the mice treated with rClone30-CD, 5-FC or saline alone, respectively. In this study, NDV was used for the first time to deliver the suicide gene for cancer therapy. Incorporation of the CD gene in the lentogenic NDV genome together with 5-FC significantly enhances cell death of HepG2 tumor cells in vitro, decreases tumor volume and increases survival of H22 tumor-bearing mice in vivo.