Zwitterionic nanogels of varying stiffness were prepared by tuning their cross-linking densities and reactant contents. In vivo studies of these nanogels show that softer nanogels pass through physiological barriers, especially the splenic filtration, more easily than their stiffer counterparts, consequently leading to longer circulation half-life and lower splenic accumulation. Results from this work emphasize the role of stiffness in designing long-circulating nanoparticles.