The canine is the most important large animal model for testing novel hemophilia A (HA) treatment. It is often necessary to use canine factor VIII (cFIII) gene or protein for the evaluation of HA treatment in the canine model. However, different biological properties between cFVIII and human FVIII (hFVIII) indicated that the development of novel HA treatment may require careful characterization of non-human FVIII. To investigate whether the data obtained using cFVIII can translate to HA treatment in human, we analyzed the differential biological properties of canine heavy chain (cHC) and light chain (cLC) by comparing with human heavy chain (hHC) and light chain (hLC). The secretion of cHC was 5-30-fold higher than hHC, with or without light chains (LCs). cHC +hLC group exhibited ˜ 18-fold increase in coagulation activity compared with hHC+hLC delivery by recombinant adeno-associated viral vectors. Unlike hHC, the secretion of cHC was independent of LCs. cLC improves the specific activity of FVIII by two-to threefold compared with hLC. Moreover, the cLC, but not cHC, contributes to the higher stability of cFVIII. Our results suggested that the cFVIII expression results in the canine model should be interpreted with caution as the cHC secreted more efficiently than hHC and cLC exhibited a more active and stable phenotype than hLC.