Human genome function is underpinned by the primary storage of genetic information in canonical B-form DNA, with a second layer of DNA structure providing regulatory control. The intercalated motif (i-Motif) DNA is a four-stranded structure built from a stack of intercalating hemiprotonated cytosine - neutral cytosine base pairs (C+:C). I-Motif structures are thought to form in cytosine-rich regions of the genome and to have regulatory functions; however, in vivo evidence for the existence of such structures has been a matter of scientific debates. Using an engineered antibody fragment (iMab), we provided strong evidence that these structures are formed in the nuclei of human cells. We demonstrated that the in vivo formation of such structures is cell-cycle and pH dependent. Furthermore, we provide evidence that i-Motif structures are formed in regulatory regions of the human genome, including promoters and telomeric regions1. Recently, we have been able to pull-down and sequence potential i-Motif forming sequences in the human genome. Our results demonstrate that the distribution of such sequences in the genome is not random. They are enriched or depleted in certain regulatory regions which might explain their potential regulatory functions. Our findings provide a core foundation for future studies exploring the biological role of this common genomic DNA structure, and for validation as a therapeutic target.