It was previously demonstrated that an intein-catalyzed splicing of B-domain deleted coagulation factor Ⅷ (BDD-FⅧ) heavy and light chains could improve the secretion of heavy chain in cis and the splicing can occur independently of cellular entities exhibiting a splicing activity in and out of the cell. In order to improve the efficacy of intein-based dual-vector delivery of the BDD-FⅧ gene, here an additional acidic region-3 (AR-3) between Pro¹⁶⁴⁰ and Ser¹⁶⁹⁰ of FⅧ proven to be helpful for the secretion of heavy chain was incorporated into BDD-FⅧ heavy chain to examine its effect on secretion and bioactivity of an intein-spliced BDD-FⅧ protein. By co-transfection of the cultured HEK293 cells with genes of the ar-3 incorporated heavy chain and light chain with fused intein (HCAR3IntN and IntCLC), an ELISA and Coatest assay were performed to determine the amount of spliced BDD-FⅧ protein and coagulation activity secreted in the culture supernatant, and the intracellular BDD-FⅧ splicing was observed by Western blotting. The data showed that the amount of spliced BDD-FⅧ protein (173±26) μg/L and coagulation activity (1.31±0.15) U/ml of supernatant from gene co-transfected cells were greater than supernatant from intein-fused ar-3-free heavy and light chain genes (HCIntN and IntCLC) co-transfected cells (102 ± 12) μg/L and (0.79 ± 0.09) U/ml indicating the additional AR-3 in the heavy chain could dramatically improve the secretion and activity of intein spliced BDD-FⅧ. A spliced BDD-FⅧ protein ((35±7) μg/L) and coagulation activity ((0.28±0.08) U/ml) was also detected in the culture supernatant of combined cells separately transfected with the HCAR3IntN and IntCLC genes implying the cellular entities independent BDD-FⅧ splicing of the intein. The total protein from gene co-transfected cells displayed an obvious protein band of the spliced BDD-FⅧ detected by a FⅧ polyclone antibody indicating the intracellular BDD-FⅧ splicing. It paved a way for ongoing study on protein trans-splicing based dual-adeno-associated virus (AAV) delivery of the split BDD-FⅧ gene in gene therapy for hemophilia A animals.