Juvenile dermatomyositis (JDM) is a debilitating pediatric autoimmune disease manifesting with characteristic rash and muscle weakness. To delineate signaling abnormalities in JDM, mass cytometry was performed with PBMCs from treatment-naive JDM patients and controls. NK cell percentages were lower while frequencies of naive B cells and naive CD4+ T cells were higher in JDM patients than in controls. These cell frequency differences were attenuated with cessation of active disease. A large number of signaling differences were identified in treatment-naive JDM patients compared with controls. Classification models incorporating feature selection demonstrated that differences in phospholipase Cγ2 (PLCγ2) phosphorylation comprised 10 of 12 features (i.e., phosphoprotein in a specific immune cell subset) distinguishing the 2 groups. Because NK cells represented 5 of these 12 features, further studies focused on the PLCγ2 pathway in NK cells, which is responsible for stimulating calcium flux and cytotoxic granule movement. No differences were detected in upstream signaling or total PLCγ2 protein levels. Hypophosphorylation of PLCγ2 and downstream mitogen-activated protein kinase-activated protein kinase 2 were partially attenuated with cessation of active disease. PLCγ2 hypophosphorylation in treatment-naive JDM patients resulted in decreased calcium flux. The identification of dysregulation of PLCγ2 phosphorylation and decreased calcium flux in NK cells provides potential mechanistic insight into JDM pathogenesis.Keywords: Autoimmunity; Calcium signaling; Immunology; Innate immunity; Rheumatology.