Surface-modified PAMAM dendrimers have important applications in drug delivery, yet a gap remains about the role that surface functionalization plays on their cell internalization capacity. We examined the cell internalization kinetics of PAMAM dendrimers that were surface-modified with acetyl, folate and poly(ethylene glycol), as model functional groups differing in size, charge, and chemical functionality. Dendrimers with 25% functionalization were internalized by HEK cells, but with slower rates and lower maximum uptakes than the native dendrimer between 1-6 h of incubation. Dendrimers with 50% functionalization exhibited negligible internalization capacities at all incubation times. Molecular dynamics simulations revealed that the solvent accessibility of the cationic surface charges is a key factor affecting cell internalization, unlike the total charge, functionality or size of surface-modified PAMAM dendrimers. These findings provide valuable insights to assist the design of PAMAM-based systems for drug delivery applications.