Toll-like receptor 9 (TLR9) plays a fundamental role in innate immune responses through pathogen-associated and danger-associated molecular pattern recognition. Ligand recognition by TLR9 results in activation of several signaling pathways, including those involving nuclear factor-kappa B, mitogen-activated protein kinases, and interfer-on-regulatory factors, which promote secretion of proinflammatory cytokines and type I interferons. TLR9 is expressed by immune-mediated cells and in clinical specimens and cell lines of various human cancers. TLR9 appears to act as a double-edged sword in cancer, with some studies indicating that it is associated with increased malignancy and others indicating that it contributes to immune response against cancer. At present, the mechanisms underlying the role of TLR9 in cancer pathophysiology are not completely clear, although various TLR9 agonists and antagonists are being tested in in vitro and in vivo cancer models as well as clinical trials. This review summarizes the current state of knowledge regarding TLR9 features, isoforms, structure, ligands, and signaling, and discusses the roles of TLR9 in cancer pathogenesis. Recent efforts to utilize TLR9 agonists and antagonists as potential anticancer immunotherapy agents are also highlighted.