Pemphigoid is a common autoimmune blistering disorder in which autoantibodies target transmembrane collagen XVII (COL17), a component of hemidesmosomes in basal keratinocytes. The ectodomain of COL17 can be cleaved from the cell surface within the juxtamembranous extracellular NC16A domain, and, interestingly, certain autoantibodies of pemphigoid patients preferentially react with the shed ectodomain. These findings suggest that COL17 ectodomain shedding generates neoepitopes on the shed form; however, the regulatory mechanism of the shedding in in vivo skin and the pathogenicity of the neoepitope-targeting antibodies still are uncertain. To address these issues, we produced rabbit antibodies specifically reacting with N-terminal cleavage sites of the shed COL17 ectodomain. The antibodies showed that certain amounts of the human COL17 ectodomain are cleaved physiologically at Gln(525) in in vivo skin. In contrast, migrating human keratinocytes cleave COL17 at Leu(524) but not at Gln(525). The passive transfer of antibodies reacting with an N-terminal cleavage site of the mouse COL17 ectodomain into neonatal wild-type mice failed to induce blister formation, even though the antibodies bound to the dermal-epidermal junctions, indicating that cleavage site-specific antibodies have reduced or absent pathogenicity for blister formation. This study shows the ectodomain shedding of COL17 to be a physiological event in in vivo human skin that probably generates nonpathologic epitopes on the cleavage sites.