BackgroundHashimoto’s thyroiditis (HT) is an organ-specific autoimmune disease characterized by lymphocyte infiltration that destroys thyrocyte cells. The aim of the present study was to elucidate the role and mechanisms of tissue small extracellular vesicle (sEV) microRNAs (miRNAs) in the pathogenesis of HT.MethodsDifferentially expressed tissue sEV miRNAs were identified between HT tissue and normal tissue by RNA sequencing in the testing set (n= 20). Subsequently, using quantitative real-time polymerase chain reaction (qRT‒PCR) assays and logistic regression analysis in the validation set (n= 60), the most relevant tissue sEV miRNAs to HT were verified. The parental and recipient cells of that tissue sEV miRNA were then explored. In vitro and in vivo experiments were further performed to elucidate the function and potential mechanisms of sEV miRNAs that contribute to the development of HT.ResultsWe identified that miR-142-3p encapsulated in T lymphocyte-derived tissue sEVs can induce Treg function defect and thyrocyte destruction through an intact response loop. Inactivation of miR-142-3p can effectively protect non-obese diabetic (NOD).H-2h4mice from HT development display reduced lymphocyte infiltration, lower antibody titers, and higher Treg cells. Looking at the mechanisms underlying sEV action on thyrocyte destruction, we found that the strong deleterious effect mediated by tissue sEV miR-142-3p is due to its ability to block the activation of the ERK1/2 signaling pathway by downregulatingRAC1.ConclusionsOur findings highlight the fact that tissue sEV-mediated miR-142-3p transfer can serve as a communication mode between T lymphocytes and thyrocyte cells in HT, favoring the progression of HT.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12916-023-02914-7.