Objective To rescue a novel recombinant oncolytic influenza virus carrying OX40 ligand and evaluate its oncolytic efficacy in hepatocellular carcinoma.Methods The gene of encoding OX40L was optimized and inserted into the NS fragment of influenza virus A/PuertoRico/8/34 (PR8). Then, the recombinant plasmid along with the remaining 7 plasmids pHW191-PB2, pHW192-PB1, pHW193-PA, pHW194-HA, pHW195-NP, pHW196-NA and pHW197-M of influenza virus PR8, were co-transfected into COSⅠ/ MDCK cells. A recombinant oncolytic influenza virus, named rFlu-OX40L, was successfully generated using reverse genetics (RG). The titers of rFlu-OX40L were determined with hemagglutination and TCID₅₀ methods. We observed the morphology and size distribution of rFlu-OX40L under electron microscopy. The growth curve of rFlu-OX40L was detected on MDCK cells. Cell viability of rFlu-OX40L was examined on various hepatoma cells with MTS assay. Cell death patterns induced by the rFlu-OX40L virus was analyzed with flow cytometry. The antitumor effect of rFlu-OX40L was evaluated based on the hepatocellular carcinoma tumor-bearing mice model.Results The oncolytic influenza virus rFlu-OX40L could be stably at the HA titer of 2^7－8 after passage in chicken embryos. The viral titers were 7-8 LgTCID₅₀ /ml. The growth curve of rFlu-OX40L was consistent with that of wild-type PR8 virus and reached the peak values at 72 h. MTS assays revealed that rFlu-OX40L at dose of 3 MOI could significantly reduce the cell viability on HCC cells, without destroying normal cells in a time and dose-dependent manner. Flow cytometry results displayed rFlu-OX40L at dose of 3 MOI could induce cell apoptosis on HCC cells in a time- and dose-dependent manner. Compared with PR8 and PBS group, the CD3+, CD4+, CD8+, CD45+, CD69+ T cell numbers in spleen cells of mice inoculated with rFlu-OX40L increased significantly.Conclusion The recombinant oncolytic influenza virus rFlu-OX40L carrying OX40L could selectively destroy hepatocellular carcinoma cells in vivo and in vitro, which will provide a novel immunotherapy strategy for clinical management of hepatocellular carcinoma.