Hyposalivation resulting from salivary gland dysfunction leads to poor oral health and greatly reduces the quality of life of patients. Current treatments for hyposalivation are limited. However, regenerative medicine to replace dysfunctional salivary glands represents a revolutionary approach. The ability of dispersed salivary epithelial cells or salivary gland-derived progenitor cells to self-organize into acinar-like spheres or branching structures that mimic the native tissue holds promise for cell-based reconstitution of a functional salivary gland. However, the mechanisms involved in salivary epithelial cell aggregation and tissue reconstitution are not fully understood. This study investigated the role of the P2Y₂ nucleotide receptor (P2Y₂R), a G protein-coupled receptor that is upregulated following salivary gland damage and disease, in salivary gland reconstitution. In vitro results with the rat parotid acinar Par-C10 cell line indicate that P2Y₂R activation with the selective agonist UTP enhances the self-organization of dispersed salivary epithelial cells into acinar-like spheres. Other results indicate that the P2Y₂R-mediated response is dependent on EGF receptor activation via the metalloproteases ADAM10/ADAM17 or the α5β1 integrin/Cdc42 signaling pathway, which leads to activation of the MAPKs JNK and ERK1/2. Ex vivo data using primary submandibular gland (SMG) cells from wild-type and P2Y₂R^-/- mice confirmed that UTP-induced migratory responses required for acinar cell self-organization are mediated by the P2Y₂R. Overall, this study suggests that the P2Y₂R is a promising target for salivary gland reconstitution and identifies the involvement of two novel components of the P2Y₂R signaling cascade in salivary epithelial cells, the α5β1 integrin and the Rho GTPase Cdc42.