Although two chain transfering separately could be used to overcome the volume limitation of adeno-associated virus vectors (AAV) in coagulation factor VIII (FVIII) gene delivery, it leads to chain imbalance for inefficient heavy chain secretion.  In this study we aimed to improve the efficacy of two chain strategy in FVIII gene delivery through the degradation of glucose-regulated protein 78 (GRP78) known as a protein chaperone in endoplasmic reticulum (ER) by deoxynivalenol (DON) to decrease GRP78-bound FVIII heavy chain.  By treating the two-chain gene transduced 293 cells with DON, the heavy chain (HC) secretion and FVIII bioactivity were observed.  Data showed that 293 cells after three hours post-treatment with DON at a concentration of 500 ng·mL⁻¹ resulted in obvious decrease the level of GRP78 but no effect on the cell proliferation.  The HC secreted from DON-treated cells transfected with HC gene alone was 59 ± 11 ng·mL⁻¹, higher than that secreted by control cells (15 ± 4 ng·mL⁻¹), and the HC secretion was further increasing to 146 ± 34 ng·mL⁻¹ in light chain (LC) gene co-transfected cells with an activity measured up to 0.66 ± 0.15 U·mL⁻¹, also greater than control cells (76 ± 17 ng·mL⁻¹ and 0.35 ± 0.09 U·mL⁻¹).  Taken together, these data suggest that DON-mediated GRP78 down-regulation could improve the efficacy of two-chain FVIII gene transfering by facilitating HC secretion, providing an experimental basis for in vivo dual-AAV application in FVIII gene delivery.