In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing.  Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding.  The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system.  After transient expression, the intracellular BDD-FVIII splicing was observed and the spliced BDD-FVIII and bioactivity secreted to culture media were quantitatively analyzed.  An enhanced splicing of BDD-FVIII with decreased protein precursors from gene co-transfected cells was observed by Western blotting.  The amount of spliced BDD-FVIII and bioactivity secreted to the culture media were 106 ± 12 ng·mL⁻¹ and 0.89 ± 0.11 U·mL⁻¹ analyzed by ELISA and Coatest method respectively, which was greater than leucine zipper free intein-fused heavy and light chain genes co-transfected cells (72 ± 10 ng·mL⁻¹ and 0.62 ± 0.07 U·mL⁻¹).  The activity of cellular mechanism-independent protein splicing was also improved, as showed by the increasing of spliced BDD-FVIII and bioactivity in culture media from combined cells separately transfected with heavy and light chain genes which was 36 ± 11 ng·mL⁻¹ and 0.28 ± 0.09 U·mL⁻¹.  It demonstrated that the leucine zippers could be used to increase the efficiency of protein trans-splicing to improve the efficacy of a dual-vector mediated BDD-FVIII gene delivery by strengthening the interaction between the two intein-pieces fused to heavy and light chains.  It provided evidence for further study in animal model using a dual-adeno-associated virus vector to deliver FVIII gene in vivo.