Extracellular vesicles (EVs) are nanoscale membrane-derived vesicles that serve as intercellular messengers carrying lipids, proteins, and genetic material. Substantial evidence has shown that cancer-derived EVs, secreted by tumor cells into the blood and other bodily fluids, play a critical role in modulating the tumor microenvironment and affecting the pathogenesis of cancer. Here we demonstrate for the first time that squamous cell carcinoma (SCC) EVs were enriched with the C-terminal fragment of desmoglein 2 (Dsg2), a desmosomal cadherin often overexpressed in malignancies. Overexpression of Dsg2 increased EV release and mitogenic content including epidermal growth factor receptor and c-Src. Inhibiting ectodomain shedding of Dsg2 with the matrix metalloproteinase inhibitor GM6001 resulted in accumulation of full-length Dsg2 in EVs and reduced EV release. When cocultured with Dsg2/green fluorescence protein–expressing SCC cells, green fluorescence protein signal was detected by fluorescence-activated cell sorting analysis in the CD90+ fibroblasts. Furthermore, SCC EVs activated Erk1/2 and Akt signaling and enhanced fibroblast cell proliferation. In vivo, Dsg2 was highly up-regulated in the head and neck SCCs, and EVs isolated from sera of patients with SCC were enriched in Dsg2 C-terminal fragment and epidermal growth factor receptor. This study defines a mechanism by which Dsg2 expression in cancer cells can modulate the tumor microenvironment, a step critical for tumor progression.—Overmiller, AM, Pierluissi, JA, Wermuth, PJ, Sauma, S., Martinez-Outschoorn, U., Tuluc, M., Luginbuhl, A., Curry, J., Harshyne, LA, Wahl, JK III, South, AP, Mahoney, MG Desmoglein 2 modulates extracellular vesicle release from squamous cell carcinoma keratinocytes.