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I-motifs are non-canonical, four-stranded DNA structures in cytosine-rich genomic regions, yet their protein-mediated regulation remains underexplored. Here, we identify PCBP1 (Poly(rC)-binding protein 1) as a selective i-motif-binding protein that unfolds specific i-motifs depending on their protonation and hairpin-forming propensities. Systematic truncation reveals that individual K-homology (KH) domains of PCBP1 cannot selectively bind or unfold i-motifs, but their coordinated actions restore wild-type PCBP1 functions. Using biochemical, biophysical, and molecular dynamics studies, we demonstrate that KH1+2 domains remodel i-motifs, recruiting KH3 to facilitate unfolding and efficient DNA replication. Chromatin and cell-based investigations reveal that PCBP1-knockdown increases i-motif formation at specific genomic loci, coinciding with G₁/S arrest and elevated ϒH2AX, indicative of genomic instability. During G₁/S transition, PCBP1 occupancy peaks at these i-motif loci, ensuring i-motif resolution in early S phase. These findings establish PCBP1 as a critical regulator of i-motif dynamics, directly linking its unfolding activity to G₁/S transition and genome stability.