In this study, a new method of cryopreservation of lactic acid bacteria (LAB) using natural deep eutectic solvents (NADESs) was developed. Survival rates of LAB during 24-h short-term and 180-day long-term cryostorage at - 20 A degrees C were investigated. The results revealed that survival of Streptococcus thermophilus (S. thermophilus) in NADESs after 24 h of cryostorage was superior to survival of two other tested LAB. Moreover, survival was higher at a ratio of NADESs to S. thermophilus of 1:1 (v/v) than observed using a 4:1 (v/v) ratio. Representative freezing characteristics of five NADESs were elucidated, including thermodynamic properties and hydrogen bonding interactions after addition of water. In order to identify the protective mechanism of NADESs on cell structure and vital metabolic enzymes of S. thermophilus during cryostorage, transmission electron microscopy (TEM) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were employed and enzyme activities of both lactate dehydrogenase (LDH) and beta-galactosidase were determined. Subsequently, NADES GlyP prepared from glycerol and L-proline was demonstrated to maintain cell membrane structural integrity and significantly (p < 0.05) maintain activities of both intracellular enzymes of S. thermophilus. Moreover, NADESs could efficiently penetrate S. thermophilus cells and intracellular beta-galactosidase activity could be used to demonstrate NADESs effectiveness in maintaining S. thermophilus survival after long-term cryostorage. These results demonstrate that NADESs can be used as green cryoprotective chemical agents (CPAs) that can efficiently increase LAB viability during cryostorage. These results should have great value in the food production and probiotics industries.