We recently demonstrated that an intein-mediated protein splicing can be used to transfer B-domain-deleted FVIII (BDD-FVIII) gene by a dual-vector.  In this study, we observed the effect of a variant heavy chain with six potential glycosylation sites of B domain and L303E/F309S mutations in its A1 domain, which were proven to be beneficial for FVIII secretion, on secretion of spliced BDD-FVIII.  By transient co-transfection of cultured 293 cells with intein-fused variant heavy chain (DMN6HCIntN) and light chain (IntCLC) genes, the culture supernatant was analyzed quantitatively by ELISA for secreted spliced BDD-FVIII antigen and by a chromogenic assay for bioactivity.  The data showed that the amount of spliced BDD-FVIII protein and coagulation activity in culture supernatant from DMN6HCIntN plus IntCLC co-transfected cells  were up to (149 ± 23) ng·mL⁻¹ and (1.12 ± 0.14) u·mL⁻¹ respectively greater than that of intein-fused wild type heavy (HCIntN) and light chain (IntCLC) co-transfected cells (99 ± 14) ng·mL⁻¹ and (0.77 ± 0.13) u·mL⁻¹   indicating that the variant heavy chain is able to improve the secretion of spliced BDD-FVIII and activity.  A cellular mechanism-independent BDD-FVIII splicing was also observed.  It provided evidence for ongoing animal experiment using intein-mediated dual-AAV vector technology for delivery of the BDD-FVIII genes.