Sjӧgren’s syndrome (SS) is a systemic autoimmune disease that targets the exocrine glands, resulting in impaired saliva and tear secretion. To date, type I interferons (IFNs) are increasingly recognized as pivotal mediators in SS, but their endogenous drivers have not been elucidated. Here, we investigate the role of mitochondrial double-stranded RNAs (mt-dsRNAs) in regulating type I IFN response in SS. We find that mt-dsRNAs are elevated in the saliva and tear of SS patients and in salivary glands of non-obese diabetic mice with salivary dysfunction. Using the in-house-developed 3D culture, we show that dsRNA stimulation increases mt-dsRNAs expression via JAK1/STAT pathway and facilitates their cytosolic export, which is accompanied by autoimmune signatures observed in SS. We further show that muscarinic receptor ligand acetylcholine or antioxidant resveratrol ameliorates autoimmune characteristics by preventing mt-dsRNA-mediated immune activation. In addition, direct suppression of mt-dsRNAs reverses the autoimmune signatures of SS. Altogether, our study underscores the significance of mt-dsRNA upregulation and suggests mt-dsRNAs as an important key to the puzzle of SS.