Microbial sensing mediated by pattern recognition receptors (PRRs) is the first key step to trigger innate immune responses, represented by the induction of type I interferons (IFNs), proinflammatory cytokines and chemokines. This innate signaling elicits an efficient activation of more specific responses in adaptive immunity. Such coordinated responses in the two systems are essential for the optimal elimination of invading microbes. Despite a major advance in our understanding of RNA sensors, TLR9 remained the only known sensor of DNA. On the other hand, there has been accumulating evidence supporting the existence of TLR9-independent DNA recognition mechanism. In this regard, DAI (also termed as DLM-1/ZBP1), the first sensor of cytosolic DNA, has recently been identified with its activation of IFN-regulatory factors(IRFs) and NF-kappaB transcriptional factors. Several recent papers suggest the involvement of an additional cytosolic DNA sensor(s). There is also a recent report that cytosolic microbial and host DNA can trigger pro-inflammatory responses via the TLR- and IRF-indepnedent pathway mediated by the inflammasome, which is consisted of NLR family members together with the adaptor protein ASC and caspase-1. In addition, evidence has been provided that host- and virus-derived proteins, which contain DNA-binding motifs (Zalpha and/or Zbeta) similar to those of DAI(DLM-1/ZBP1), negatively regulates the immune response that is activated by cytosolic DNA. Thus, these recent findings reveal the complex DNA-sensing mechanism for triggering the activation of innate immunity, and the breakdown of this sensing mechanism may lead to autoimmune abnormalities.