Human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (hiPSC-RPE) cells suspension have the potential for regenerative treatment. However, practical regenerative applications with hiPSC-RPE cells require the development of simple and cost-effective non-freezing preservation methods. We investigated the effect of non-freezing temperatures on suspended hiPSC-RPE cells in various conditions and analysed mechanisms of cell death, anoikis, Rho GTPases, hypoxia, microtubule destruction, and cell metabolism. Cells stored at 37 °C had the lowest viability due to hypoxia from high cell metabolism and cell deposits, and cells preserved at 4 °C were damaged via microtubule fragility. Cell suspensions at 16 °C were optimal with drastically reduced apoptosis and negligible necrosis. Moreover, surviving cells proliferated and secreted key proteins normally, compared to cells without preservation. hiPSC-RPE cell suspensions were optimally preserved at 16 °C. Temperatures above or below the optimal temperature decreased cell viability significantly yet differentially by mechanisms of cell death, cellular metabolism, microtubule destruction, and oxygen tension, all relevant to cell conditions. Surviving cells are expected to function as grafts where high cell death is often reported. This study provides new insight into various non-freezing temperature effects on hiPSC-RPE cells that are highly relevant to clinical applications and may improve cooperation between laboratories and hospitals.