Self-assembled nanogels made of hydrophobized pullulan were obtained using a versatile, simple, reproducible and low-cost method. In a first reaction pullulan was modified with hydroxyethyl methacrylate or vinyl methacrylate, further modified in the second step with hydrophobic 1-hexadecanethiol, resulting as an amphiphilic material, which self-assembles in water via the hydrophobic interaction among alkyl chains. Structural features, size, shape, surface charge and stability of the nanogels were studied using hydrogen nuclear magnetic resonance, fluorescence spectroscopy, cryo-field emission scanning electron microscopy and dynamic light scattering. Above the critical aggregation concentration spherical polydisperse macromolecular micelles revealed long-term colloidal stability in aqueous medium, with a nearly neutral negative surface charge and mean hydrodynamic diameter in the range 100–400 nm, depending on the polymer degree of substitution. Good size stability was observed when nanogels were exposed to potential destabilizing pH conditions. While the size stability of the nanogel made of pullulan with vinyl methacrylate and more hydrophobic chains grafted was affected by the ionic strength and urea, nanogel made of pullulan with hydroxyethyl methacrylate and fewer hydrophobic chains grafted remained stable.