Gibbon ape leukemia virus (GALV) can infect a wide variety of cells but fails to infect most cells derived from laboratory mice. Transduction of human hematopoietic stem cells with GALV retroviral vectors is more efficient than with amphotropic vectors. In this study, a Moloney murine leukemia virus-gibbon ape leukemia virus (MoMLV-GALV) vector was constructed by replacing the natural env gene of the full-length Moloney MLV genome with the GALV env gene. To monitor viral transmission by green fluorescent protein (GFP) expression, internal ribosomal entry site-enhanced GFP (IRES-EGFP) was positioned between the GALV env gene and the 3′ untranslated region (3′ UTR) to obtain pMoMLV-GALV-EGFP. The MoMLV-GALV-EGFP vector was able to replicate with high titer in TE671 human rhabdomyosarcoma cells and U-87 human glioma cells. To evaluate the potential of the MoMLV-GALV vector as a therapeutic agent, the gene for the fusogenic envelope G glycoprotein of vesicular stomatitis virus (VSV-G) was incorporated into the vector. Infection with the resulting MoMLV-GALV-VSV-G vector resulted in lysis of the U-87 cells due to syncytium formation. Syncytium formation was also observed in the transfected human prostate cancer cell line LNCaP after extended cultivation of cells. In addition, we deleted the GALV env gene from the MoMLV-GALV-VSV-G vector to improve viral genome stability. This MoMLV-VSV-G vector is also replication competent and induces syncytium formation in 293T, HT1080, TE671 and U-87 cells. These results suggest that replication of the MoMLV-GALV-VSV-G vector or MoMLV-VSV-G vector may directly lead to cytotoxicity. Therefore, the vectors developed in this study are potentially useful tools for cancer gene therapy.