DNase II digests DNA in endolysosomes. In the absence of DNase II, undigested DNA activates cytoplasmic DNA-sensing pathways. Little is known, however, about the role of DNase II in endolysosomal DNA sensing by TLR9. Here we show that DNase II is required for TLR9. We test two types of TLR9 ligands, CpG-A and CpG-B, and show that only CpG-A response is impaired in DNase II-deficient dendritic cells (DCs). Enzymatically inactive DNase II mutants cannot rescue CpG-A responses. DNase II cleaves CpG-A from 20-mer to 11-12-mer. The 3 0 11-mer CpG-A fragment activates DNase II-deficient DCs. CpG-A shows higher co-localization with LAMP-2 þ lysosomes than CpG-B and induces DNase II localization in LAMP-2 þ lysosomes. Moreover, we demonstrate that DNase II is required for TLR9 activation by bacterial genomic DNA. Taken together, these results demonstrate that TLR9 responds to DNA fragments generated by DNase II.