An i-motif (iM) is a four-stranded (quadruplex) DNA structure that folds from cytosine (C)-rich sequences. iMs can fold under many different conditions in vitro, which paves the way for their formation in living cells. iMs are thought to play key roles in various DNA transactions, notably in the regulation of genome stability, gene transcription, mRNA translation, DNA replication, telomere and centromere functions, and human diseases. We summarize the different techniques used to assess the folding of iMs in vitro and provide an overview of the internal and external factors that affect their formation and stability in vivo. We describe the possible biological relevance of iMs and propose directions towards their use as target in biology.