Although DNA is most widely known for its ability to store and pass along genetic information, the discovery of G-quadruplex structures has illuminated a new role for DNA in biology. DNA G-quadruplexes are four-stranded globular nucleic acid secondary structures formed in specific G-rich sequences with biological significance, such as human telomeres and oncogene promoters. This review focuses on the unimolecular DNA G-quadruplexes, which can readily form in solution under physiological conditions and are considered to be the most biologically relevant. Available structural data show a great conformational diversity of unimolecular G-quadruplexes, which are amenable to small-molecule drug targeting. The relationships between sequence, structure, and stability of unimolecular DNA G-quadruplexes, as well as the recent progress on interactions with small-molecule compounds and insights into rational design of G-quadruplex-interactive molecules, will be discussed.