Introduction:　Antifreeze　peptides　regulate　the　physiological　functions　of　frozen　cells　and　even　their　apop-tosis;　however,　the　mechanisms　by　which　antifreeze　peptides　regulate　these　processes　remain　unclear,　although　the　interactions　between　cell　membranes　and　ice　are　well　known　to　be　important　in　this　pro-cess.Objectives:　Our　study　aims　to　investigate　how　antifreeze　peptides　regulate　cell　physiological　functions　during　the　freezing　process.　Methods:　We　investigated　the　cryoprotective　effect　of　rsfAFP　on　the　physiological　functions　of　S.　ther-mophilus　under　freezing　stress　by　measuring　cellular　metabolism　activity,　intracellular　enzyme　activity,　cell　membrane　characterization,　and　cell　apoptosis.　The　mechanism　by　which　rsfAFP　impacts　S.　ther-mophilus　physiological　functions　under　freezing　stress　was　investigated　using　multispectral　techniques　and　cryo-TEM.Results:　We　show　that　a　recombinant　antifreeze　peptide　(rsfAFP)　interacts　with　the　extracellular　capsular　polysaccharides　and　peptidoglycan　of　Streptococcus　thermophilus　and　ice　to　cover　the　outer　layer　of　the　membrane,　forming　a　dense　protective　layer　that　regulates　the　molecular　structure　of　extracellular　ice　crystals,　which　results　in　reduced　extracellular　membrane　damage,　depressed　apoptosis　and　increased　intracellular　metabolic　activity.　This　interaction　mechanism　was　indicated　by　the　fact　that　S.　thermophilus　better　maintained　its　permeability　barrier,　membrane　fluidity,　membrane　structural　integrity,　and　cytoplasmic　membrane　potential　during　freezing　stress　with　rsfAFP　treatment.Conclusion:　These　results　provide　new　insights　into　the　mechanism　by　which　rsfAFP　regulates　frozen　cell　physiological　functions　and　apoptosis　under　freezing　stress.(c)　2023　The　Authors.　Published　by　Elsevier　B.V.　on　behalf　of　Cairo　University.　This　is　an　open　access　article