Toll-like receptor 9 (TLR9) is specialized for the recognition of pathogenic nucleic acids. TLR9 is expressed in intracellular compartments where it responds specifically to pathogen DNA. Several factors contribute to the ability of TLR9 to discriminate between self and foreign DNA. Regulatory mechanisms of the innate and adaptive immune system exist that balance the immune responses mediated by TLR9. Short synthetic CpG oligodeoxynucleotides are used to induce controlled and directed TLR9-dependent stimulation and are effective immune modulators in preclinical and clinical studies. This review will summarize the interplay between TLR9-dependent opposing stimulatory and regulatory effects in innate and adaptive immunity.