Advances in stem cell technology and regenerative medicine have underscored the need for effective banking of living cells. Cryopreservation, using very low temperatures to achieve suspended animation, is widely used to store or bank cells for later use. This process requires the use of cryoprotective agents (CPAs) to protect cells against damage caused by the cooling and warming process. However, current popular CPAs like DMSO can be toxic to cells and must be thoroughly removed from cells before they can be used for research or clinical applications. Trehalose, a nontoxic sugar found in organisms capable of withstanding extreme cold or desiccation, has been explored as an alternative CPA. The disaccharide must be present on both sides of the cellular membrane to provide cryo-protection. However, trehalose is not synthesized by mammalian cells nor has the capability to diffuse through their plasma membranes. Therefore, it is crucial to achieve intracellular delivery of trehalose for utilizing the full potential of the sugar for cell banking. In this review, various methods that have been explored to deliver trehalose into mammalian cells for their banking at both cryogenic and ambient temperatures are surveyed. Among them, the nanoparticle-mediated approach is particularly exciting. Collectively, studies in the literature demonstrate the great potential of using trehalose as the sole CPA for cell banking, to facilitate the widespread use of living cells in modern medicine.