Lyophilization of human red blood cells has important significance in clinical application. Some sugars, especially trehalose, can be more tolerant of some organism or cells to dry environments, But, how to bring sugars into cells is a challenge. This study was aimed to investigate the regularity of sugar-uptake in human red blood cells. The absorption rate of trehalose and glucose in red blood cells, free hemoglobin level and erythrocyte deformation index were determined at different incubation temperature (4, 25 and 37 degrees C), different sugar concentration (0, 0.2, 0.4, 0.6, 0.8 and 1 mol/L) and different incubation time (1, 3, 5, 7 and 9 hours). The results showed that with increase of temperature and extracellular sugar concentration, the uptake of sugar in red blood cells also increased, the intracellular trehalose and glucose concentrations were over 30 mmol/L and 40 mmol/L respectively. The effects of incubation time on uptake of trehalose and glucose were different. With prolonging of incubation time, the uptake of trehalose showed firstly increase and then decrease, however, the uptake of glucose showed a constant increase. But the loading process had side-effect on free hemoglobin and maximum deformation index (MAXDI) of red blood cells, especially for trehalose, which mainly come from high osmotic pressure. It is concluded that the uptake of sugars in red blood cells is closely dependent on incubation temperature, extracellular sugar concentration and incubation time. In certain condition, the efficiency of sugar uptake is very high, but this process also damages red blood cells so as to affect the application of sugars in lyophilization of red blood cells. The research in the future should focus on how to deal with the relation between cell injury and uptake efficiency of sugar in red blood cells.