i-Motif (the name stems from “intercalated”), also known as i-DNA, is a pH-dependent four-stranded nucleic acid structure formed by cytosine-rich sequences via hemi-protonated and intercalated C
C+ base pairs. Although this structure is favored at acidic pH, recent evidence has demonstrated its existence in vivo, stimulating the exploration of its biological roles. Before that, it was mostly regarded as a mere structural oddity, or a tool for bio- and nanotechnol- ogies: its unique pH-sensitive nature makes it a remarkable candidate as a nanodevice and pH sensor. In this chapter, we provide a general panorama of this structure. The history and basic knowledge of i-motif are provided first. Then, we present the main characterization methods of i-motif and factors affecting i-motif stability. Following that, we focus on the applications of i-motif in nanotechnology and analytical chemistry. Last, the interaction between i-motif and ligands and the physiological roles of i-motif are briefly introduced. We argue that the i-motif, similar to its complementary G-quadruplex, is an attractive structure for multidisciplinary approaches. It serves as a basic component for various applications and has been proposed to play biological roles in vivo.