This study tested the hypothesis that mtDNA fragments carry immunostimulatory motifs that naturally induce immune activation by PDC. Genomic and mtDNA induced similar IFN-α production after transfection into PBMCs using the liposomal transfection reagent DOTAP. Shortening of mtDNA to CpG islands enhanced the immunostimulatory activity, based on the presence of unmethylated CpG DNA. Further fragmentation into mtODN, which exhibited similarities to published CpG ODN, resulted in a strong immunostimulatory activity in addition to PDC maturation and migration. The addition of the human cathelicidin LL-37 to CpG islands induced spontaneous PDC IFN-α production. Notably, one phosphodiester mtODN with a double-palindromic structure induced PDC IFN-α production in the absence of DOTAP. Flow cytometry, life-cell, and confocal imaging revealed attachment and spontaneous uptake into PDC, colocalizing, in part, with TLR9 in early endosomal vesicles. This process was accompanied by a moderate but significant PDC maturation in addition to B cell and NK cell activation (P<0.05). Altogether, our data indicate that fragmented mtDNA, which may be released as a consequence of apoptotic, necrotic, and necroptotic cell death, can act as a DAMP. For the first time, our study provides a mechanism how longer and shorter mtDNA fragments can be taken up naturally by the PDC and thus, may contribute to acute and chronic immune activation.