The i-motif is a pH-responsive cytosine-rich oligonucleotide sequence that forms, under acidic conditions, a quadruplex structure. This tunable structural switching has made the i-motif a useful platform for designing pH-responsive nanomaterials. Despite the widespread application of i-motif DNA constructs as biomolecular switches, the mechanism of i-motif folding on the atomic scale has yet to be established. We investigate the early folding structural dynamics of i-motif oligonucleotides with laser-pulse-induced pH-jump time-resolved X-ray solution scattering. Following the pH-jump, we observe that the initial random coil ensemble converts into a contracted intermediate state within 113 ns followed by further folding on the 10 ms time scale. We reveal the representative structures of these transient species, hitherto unknown, with molecular dynamics simulations and ensemble fitting. These results pave the way for understanding metastable conformations of i-motif folding and for benchmarking emerging theoretical models for simulating noncanonical nucleic acid structures.