Traditional　cryoprotectants　such　as　sucrose　and　phosphates　are　limited　by　safety　and　flavor　concerns,　creating　a　demand　for　food-grade　alternatives.　Here,　Bos　taurus　domesticus　gelatin　antifreeze　peptides　(BtAFP)　were　investigated　for　their　dual　cryoprotective　mechanisms.　Rheological　analysis　showed　that　BtAFP　enhanced　gel　elasticity　and　delayed　freezing,　exhibiting　superior　cryoprotection　compared　with　bovine　serum　albumin.　LC–MS/MS　identified　452　peptides,　from　which　five　nontoxic　collagen-derived　candidates　with　amphiphilic　α-helix/β-sheet　motifs　were　selected.　Ice　recrystallization　assays　showed　that　the　majority　of　ice　crystals　in　purified　peptide　solutions　were　concentrated　below　1000　μm2.　Differential　scanning　calorimetry　indicated　that　GKEAFP,　GLPAFP,　and　GLRAFP　most　effectively　inhibited　ice　growth.　Molecular　docking　demonstrated　stable　peptide–ice　and　peptide−α-lactalbumin　interactions,　with　GLPAFP　notably　stabilizing　protein　structures　through　extensive　hydrogen　bonding　and　surface　interactions.　These　findings　suggest　that　gelatin-derived　antifreeze　peptides　suppress　ice　recrystallization　and　stabilize　proteins　via　amphiphilic　ice-binding　motifs,　providing　promising　natural　cryoprotectants　for　frozen　food　applications.　©　2025　American　Chemical　Society