Cytosine (C)-rich regions of single-stranded DNA or RNA can fold into a tetraplex structure called i-motifs, which are typically stable under acidic pHs due to the need for protons to stabilize C–C interactions. While new studies have shown evidence for the formation of i-motifs at neutral and even physiological pH, it is not clear whether i-motifs can stably form in cells where DNA experiences topological constraint and crowding. Similarly, several studies have shown that a molecularly crowded environment promotes the formation of i-motifs at physiological pH; however, whether the intracellular crowding counteracts the topological destabilization of i-motifs is yet to be investigated. In this manuscript, using fluorescence resonance energy transfer (FRET)-based single-molecule analyses of human telomeric (hTel) i-motifs embedded in nanocircles as a proof-of-concept platform, we investigated the overall effects of crowding and topological constraint on the i-motif behavior. The smFRET analysis of the nanoassembly showed that the i-motif remains folded at pH 5.5 but unfolds at higher pHs. However, in the presence of a crowder (30% PEG 6000), i-motifs are formed at physiological pH overcoming the topological constraint imposed by the DNA nanocircles. Analysis of FRET-time traces show that the hTel sequence primarily assumes the folded state at pH ≤7.0 under crowding, but it undergoes slow conformational transitions between the folded and unfolded states at physiological pH. Our demonstration that the i-motif can form under cell-mimic crowding and topologically constrained environments may provide new insights into the potential biological roles of i-motifs and also into the design and development of i-motif-based biosensors, therapy, and other nanotechnological applications.