Background Mitochondrial DNA (mtDNA) is a potent activator for pro‐inflammatory response. Dendritic cells (DCs) are immunosuppressed in sepsis, whether mtDNA mediates immunoparalysis in sepsis remains unknown. Methods The mRNAs were assessed by qPCR. Flow cytometry was used to measure the expression of costimulatory molecules and the proliferation of CD4+ T cells. Western blot and immunofluorescence staining were used to analyse the expression of proteins. Cytokine secretion was detected by ELISA. Histology of lung tissue was used to assess the inflammatory injury. Results Lipopolysaccharide‐induced endotoxemia increased plasma mtDNA levels and immunoparalysis of spleen DCs, while hydrolysing mtDNA reversed immunoparalysis of spleen DCs in vivo. Moreover, cytoplasmic mtDNA of DCs was accumulated in endotoxemia and sepsis. mtDNA transfection into bone marrow‐derived DCs (BMDCs) inhibited the expression of costimulatory molecules (e.g., CD40, CD80 and CD86) and the release of IL‐12p70, while increasing the secretion of IL‐10. Cytoplasmic mtDNA also inhibited the ability of BMDCs to promote the proliferation of CD4+ T cells. Mechanistic analysis revealed that STING signalling was required for mtDNA‐mediated immunoparalysis of DCs in vivo and in vitro. Further studies showed deletion of STING reversed mtDNA‐mediated immunoparalysis of DCs and improved the prognosis of endotoxemia and sepsis. Conclusion Our results demonstrated that mtDNA promotes immunoparalysis of DCs, and contributes to sepsis‐associated immunosuppression by activating STING signalling. Our study may provide new insights to elucidate the molecular pathogenesis of immunosuppressive DCs in sepsis.