The carboxyl-terminal domains of von Willebrand factor, D4-CK, are cysteine-rich implying that they are structurally important. In this study we characterized the impact of five cysteine missense mutations residing in D4-CK domains on the conformation and biosynthesis of von Willebrand factor. These variants were identified as heterozygous in type 1 (p.Cys2619Tyr and p.Cys2676Phe), type 2A (p.Cys2085Tyr and p.Cys2327Trp) and as compound heterozygous in type 3 (p.Cys2283Arg) von Willebrand disease. Transient expression of human cell lines with wild-type or mutant von Willebrand factor constructs was performed. The mutated and wild-type recombinant von Willebrand factors were quantitatively and qualitatively assessed and compared. Storage of von Willebrand factor in pseudo-Weibel-Palade bodies was studied with confocal microscopy. The structural impact of the mutations was analyzed by homology modeling. Homozygous expressions showed that these mutations caused defects in multimerization, elongation of pseudo-Weibel-Palade bodies and secretion of von Willebrand factor. Co-expressions of wild-type von Willebrand factor and p.Cys2085Tyr, p.Cys2327Trp and p.Cys2283Arg demonstrated defective multimer assembly, suggesting a new pathological mechanism for dominant type 2A von Willebrand disease due to mutations in D4 and B domains. Structural analysis revealed that mutations p.Cys2283Arg, p.Cys2619Tyr and p.Cys2676Phe disrupted intra-domain disulfide bonds, whereas p.Cys2327Trp might affect an inter-domain disulfide bond. The p.Cys2327Trp variant is distinguished from the other mutants by an electrophoretic mobility shift of the multimer bands. The results highlight the importance of cysteine residues within the carboxyl-terminal of von Willebrand factor on structural conformation of the protein and consequently multimerization, storage, and secretion of von Willebrand factor.