We demonstrate that programmed assemblies of DNA origami have a substantial ability to protect cells undergoing freeze-thaw cycles; thereby, they can be used as cryoprotecting agents as their nanoscale morphology and ice-philicity are tailored. A single-layered DNA origami nanopatch functionalized with antifreeze threonine peptides enabled the viability of HSC-3 cells to reach 56% after 1-month cryopreservation under liquid nitrogen, surpassing dimethyl sulfoxide, which produced 38% viability. 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 impact on current cryopreservation technologies.