Cryopreservation of cells is essential for the conservation and cold chain of bioproducts and cell-based medicines. Here, we demonstrate that self-assembled DNA origami nanostructures have a substantial ability to protect cells undergoing freeze-thaw cycles; thereby, they can be used as cryoprotectant agents, because their nanoscale morphology and ice-philicity are tailored. In particular, a single-layered DNA origami nanopatch functionalized with antifreezing threonine peptides enabled the viability of HSC-3 cells to reach 56% after 1 month of cryopreservation, surpassing dimethyl sulfoxide, which produced 38% viability. It also exhibited minimal dependence on the cryopreservation period and freezing conditions. We attribute this outcome to the fact that the peptide-functionalized DNA nanopatches exert multisite actions for the retardation of ice growth in both intra- and extracellular regions and the protection of cell membranes during cryopreservation. This discovery is expected to deepen our fundamental understanding of cell survival under freezing environment and affect current cryopreservation technologies.