This article reviews the role of surface charge in the cellular uptake and cytotoxicity of medical nanoparticles (NPs). Surface charge is a critical factor influencing NP function in imaging and drug delivery. Positive charge generally improves imaging efficacy and gene transfer efficiency but increases cytotoxicity. Cationic NPs cause more severe disruption of plasma membrane integrity, mitochondrial and lysosomal damage, and an increased number of autophagosomes compared to anionic NPs. Nonphagocytic cells take up cationic NPs more efficiently, while phagocytic cells prefer anionic NPs. The different uptake preferences of phagocytic and nonphagocytic cells for cationic and anionic NPs can influence the efficacy and selectivity of NP-based drug delivery and imaging. The cytotoxicity of NPs is influenced by particle parameters such as morphology, size, shape, composition, surface charge, and surface hydrophobicity. The presence of serum can reduce NP uptake in nonphagocytic cells but increase it in phagocytic cells. The mechanisms of NP entry into cells, including passive and active transport, endocytosis, and paracellular transport, are also discussed. The article highlights the need to consider the differences between phagocytic and nonphagocytic cells when designing medical NPs to optimize their efficacy and safety.This article reviews the role of surface charge in the cellular uptake and cytotoxicity of medical nanoparticles (NPs). Surface charge is a critical factor influencing NP function in imaging and drug delivery. Positive charge generally improves imaging efficacy and gene transfer efficiency but increases cytotoxicity. Cationic NPs cause more severe disruption of plasma membrane integrity, mitochondrial and lysosomal damage, and an increased number of autophagosomes compared to anionic NPs. Nonphagocytic cells take up cationic NPs more efficiently, while phagocytic cells prefer anionic NPs. The different uptake preferences of phagocytic and nonphagocytic cells for cationic and anionic NPs can influence the efficacy and selectivity of NP-based drug delivery and imaging. The cytotoxicity of NPs is influenced by particle parameters such as morphology, size, shape, composition, surface charge, and surface hydrophobicity. The presence of serum can reduce NP uptake in nonphagocytic cells but increase it in phagocytic cells. The mechanisms of NP entry into cells, including passive and active transport, endocytosis, and paracellular transport, are also discussed. The article highlights the need to consider the differences between phagocytic and nonphagocytic cells when designing medical NPs to optimize their efficacy and safety.