von Willebrand factor (VWF) is a multimeric plasma protein that mediates platelet adhesion to sites of vascular injury. The hemostatic function of VWF depends upon the formation of disulfide-linked multimers, which requires the VWF propeptide (D1D2 domains) and adjacent D'D3 domains. VWF multimer assembly occurs in the trans-Golgi at pH ~ 6.2 but not at pH 7.4, which suggests that protonation of one or more His residues (pK(a) ~6.0) mediates the pH dependence of multimerization. Alignment of 30 vertebrate VWF sequences identified 13 highly conserved His residues in the D1D2D'D3 domains, and His-to-Ala mutagenesis identified His³⁹⁵ and His⁴⁶⁰ in the D2 domain as critical for VWF multimerization. Replacement of His³⁹⁵ with Lys or Arg prevented multimer assembly, suggesting that reversible protonation of this His residue is essential. In contrast, replacement of His⁴⁶⁰ with Lys or Arg preserved normal multimer assembly, whereas Leu, Met, and Gln did not, indicating that the function of His⁴⁶⁰ depends primarily upon the presence of a positive charge. These results suggest that pH sensing by evolutionarily conserved His residues facilitates the assembly and packaging of VWF multimers upon arrival in the trans-Golgi.