Zwitterionic nanogels of varying stiffness were prepared by tuning their cross-linking densities and reactant contents. <i>In vivo</i> 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