Molecular permeation through phospholipid membranes is a fundamental biological process for small molecules. Sucrose is one of the most widely used sweeteners and a key factor in the pathogenesis of obesity and diabetes, yet a detailed understanding of its mechanism involved in permeability into phospholipid membranes is still lacking. Here, using giant unimolecular vesicles (GUVs) reconstituting membrane properties, we compared the osmotic behavior of sucrose in GUVs and HepG2 cells to explore the effect of sucrose on membrane stability in the absence of protein enhancers. The results suggested that the particle size and potential of GUVs and the cellular membrane potential changed significantly with increasing the sucrose concentration (p < 0.05). In microscopic images of cells containing GUVs and sucrose, the fluorescence intensity of vesicles was 537 ± 17.69 after 15 min, and the value was significantly higher than that of microscopic images of cells without sucrose addition (p < 0.05). These changes suggested that the permeability of the phospholipid membrane became larger under a sucrose environment. This study provides a theoretical basis for better insight on the role of sucrose in the physiological environment.