In cryopreservation, which is an important process for the long-term storage and transport of cells, cryopreservation solutions containing cryoprotectants are usually used to protect cells from damage. However, most cryoprotectants have non-negligible cytotoxicity and side effects that greatly limit their applications in clinical use. This has led to an increased need for more biocompatible cryoprotectants, and interest in bioinspired cryoprotectants is increasing. In this study, we have discovered the potential of using natural acid-form sophorolipids (aSL) as a cryoprotectant, which suppresses ice formation and reduces osmotic stress. The solution containing aSL showed the smallest size of ice crystals compared to the solutions containing other cryoprotectants, such as dimethyl sulfoxide (Me2SO), glycerol (GL), ethylene glycol (EG), and propylene glycol (PG). By adding aSL to a hypertonic culture medium, cell viability was significantly improved. This finding suggests an opportunity to develop low-toxicity and efficient reagents for cell cryopreservation in the future.