Pemphigus vulgaris (PV) acantholysis represents a complex phenomenon wherein a number of factors cooperates. PV serum is known to modulate important cellular events, including kinase activity, transcriptional regulation, and proteinase expression. Indeed, transduction of signals to the cell triggered by PV serum may induce proteinase up-regulation potentially responsible for disruption of epidermal adhesion and, ultimately, blister formation. Here, we sought to investigate this hypothesis by using both in vivo and in vitro models of PV. Microarray analysis on mouse skin tissues suggested that the equilibrium between extracellular proteinases and their inhibitors moved towards enhanced proteolytic activity in PV neonatal mouse model, at least on the transcriptional level. Conversely, genes codifying cell adhesion proteins were dramatically down-regulated. The effects of PV serum on the protein level were then studied in vitro both in keratinocyte monolayers and skin organ cultures focusing on matrix metalloproteinase (MMP) 9 expression and activity. By means of Western blotting, zymography, and living cell immunofluorescence studies, we showed that MMP-9 was early overexpressed in keratinocytes exposed to PV serum, and subsequently secreted in the culture medium. However, we failed to demonstrate extracellular activation of MMP-9, since it was found in its 92 kDa inactive form in serum-free culture supernatants. Taken together, our data demonstrated that proteinase expression, particularly of MMP-9, is modulated by PV serum and associated with PV acantholysis.