Mitochondrial defects and antimitochondrial cardiolipin (CL) antibodies are frequently detected in autoimmune disease patients. CL from dysregulated mitochondria activates various pattern recognition receptors, such as NLRP3. However, the mechanism by which mitochondrial CL activates APCs as a damage‐associated molecular pattern to prime antigen‐specific naïve T cells, which is crucial for T‐cell‐dependent anticardiolipin IgG antibody production in autoimmune diseases is unelucidated. Here, we show that CL increases the expression of costimulatory molecules in CD11c+ APCs both in vitro and in vivo. CL activates CD11c+ APCs via TLR2‐PI3K‐PKN1‐AKT/p38MAPK‐NF‐κB signaling. CD11c+ APCs that have been activated by CL are sufficient to prime H‐Y peptide‐specific naïve CD4+ T cells and OVA‐specific naïve CD8+ T cells. TLR2 is necessary for anti‐CL IgG antibody responses in vivo. Intraperitoneal injection of CL does not activate CD11c+ APCs in CD14 KO mice to the same extent as in wild‐type mice. CL binds to CD14 (Kd = 7 × 10−7 M). CD14, but not MD2, plays a role in NF‐kB activation by CL, suggesting that CD14+ macrophages contribute to recognizing CL. In summary, CL activates signaling pathways in CD11c+ APCs through a mechanism similar to gram (+) bacteria and plays a crucial role in priming antigen‐specific naïve T cells. Cardiolipin induce cytoskeletal rearrangement and upregulate expression of costimulatory molecules of CD11c+ APCs via TLR2‐PI3K‐PKN1‐AKT/p38MAPK‐NF‐κB signaling pathway. CL induces proliferation and IFN‐γ production of antigen‐specific T cells when OVA or H‐Y peptide was administered together. CD14 also plays a role in activation of CD11c+ APCs in response to CL.