Narrowly dispersed zwitterionic poly(amido amine) (PAA) nanogels with a diameter of approximately 100 nm were prepared by a high-yielding and surfactant-free, inverse nanoprecipitation of PAA polymers. The resulting, negatively charged, nanogels (PAA-NG1) were functionalized with N,N-dimethylethylenediamine via EDC/NHS coupling chemistry. This resulted in nanogels with a positive surface charge (PAA-NG2). Both types of nanogels were fluorescently labelled via isothiocyanate coupling. PAA-NG1 displays high colloidal stability both in PBS and Fetal Bovine Serum solution. Moreover, both nanogels exhibit a distinct zwitterionic swelling profile in response to pH changes. Cellular uptake of FITC-labelled nanogels with RAW 264.7, PC-3 and COS-7 cells was evaluated by fluorescence microscopy. These studies showed that nanogel surface charge greatly influences nanogel–cell interactions. The PAA polymer and PAA-NG1 showed minimal cell toxicity as was evaluated by MTT assays. The findings reported here demonstrate that PAA nanogels possess interesting properties for future studies in both drug delivery and imaging.