Sugar plays a vital role in the structural and functional characteristics of cells. Hence, the interaction of NPs with cell membranes in the presence of sugar concentrations is important for medicinal and pharmacological innovations. This study integrated three tools: giant unilamellar vesicles (GUVs), anionic magnetite nanoparticles (NPs), and sugar concentrations, to understand a simplified mechanism for interactions between the vesicle membranes and NPs under various sugar concentrations. We focused on changing the sugar concentration in aqueous solution; more precisely, sucrose inside the GUVs and glucose outside with equal osmolarity. 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were used to prepare the charged membranes of 40mole%DOPG/60mole%DOPC-GUVs, whereas only DOPC was used to prepare the neutral membranes. Phase contrast fluorescence microscopy shows that the adherence of 18 nm magnetite NPs with anionic charge depends on the sugar concentration. The alterations of GUVs induced by the NPs are characterized in terms of i) vesicle compactness, ii) deformation, and iii) membrane poration. The presence of sugar provides additional structural stability to the GUVs and reduces the effects of the NPs with respect to these parameters; more precisely, the higher the sugar concentration, the smaller the alteration induced by the NPs. The differences in NPs effects are explained by the change in the type of interaction between sugar molecules and lipid membranes, namely enthalpy and entropy-driven interaction, respectively. In addition, such alterations are influenced by the surface charge density of the lipid bilayer. The surface pressure of membranes due to the adsorption of NPs is responsible for inducing the poration in membranes. The differences in deformation and poration in charged and neutral GUVs under various sugar concentrations are discussed based on the structure of the head of lipid molecules.