von Willebrand factor (vWF) is a huge multimeric plasma glycoprotein with important functions involved in thrombosis and hemostasis. Its qualitative and/or quantitative abnormalities result in a bleeding disorder termed von Willebrand disease (VWD). Gene therapy is favorable for treatment of VWD because this disease is monogenic and the vWF is a secretory protein making non-specific targeting organ required for gene delivery. But the vWF gene is hardly packaged in most existing viral vectors especially the desired adeno- associated virus (AAV) vectors for its oversized cDNA in size (8.4 kb). The intein-mediated protein trans-splicing was explored to co-transfer split three fragmented vWF gene into eukaryotic cells by a ternary-vector system and the functional vWF protein was expected to be formed posttranslationally by protein trans-splicing. The vWF cDNA was broken into three fragments before codons of Cys¹⁰⁹⁹ and Ser²⁰⁰⁴ which required for protein splicing and then fused with Ssp DnaE and Ssp DnaB inteins respectively. A group of three eukaryotic expression vectors were produced by inserting these three fusion genes into pcDNA3.1(+) respectively. By transient co-transfection of 293 cells with these three vectors the conditioned culture supernatant was observed for vWF multimer pattern by electrophoresis, analyzed for vWF antigen and binding capacity of coagulation factor Ⅷ (FⅧ) quantitatively. With FⅧ gene co-transfection, the antigen and activity of FⅧ in the culture supernatant were measured. The data showed that with intein-mediated protein trans-splicing after translation the culture supernatant from cells co-transfected with intein-fused three fragmented vWF genes displayed a vWF multimer pattern and FⅧ binding capacity similar to normal human plasma and cells of vWF gene transfected positive control, and the FⅧ secretion and activity were increased dramatically in FⅧ gene co-transfected cells indicating the functional recovery of spliced vWF as a FⅧ carrier. It suggests that inteins could be used as a powerful means for tri-fragmental co-delivery of the vWF gene and may be valuable for application of intein-based ternary AAV vector in split vWF gene delivery in gene therapy for VWD to overcome the packaging limitation of AAV vectors.