Amphiphilic mannan (mannan-C(16)) was synthesized by the Michael addition of hydrophobic 1-hexadecanethiol (C(16)) to hydroxyethyl methacrylated mannan (mannan-HEMA). Mannan-C(16) formed nanosized aggregates in water by self-assembly via the hydrophobic interaction among C(16) molecules as confirmed by hydrogen nuclear magnetic resonance ((1)H NMR), fluorescence spectroscopy, cryo-field emission scanning electron microscopy (cryo-FESEM), and dynamic light scattering (DLS). The mannan-C(16) critical aggregation concentration (cac), calculated by fluorescence spectroscopy with Nile red and pyrene, ranged between 0.04 and 0.02 mg/mL depending on the polymer degree of substitution of C(16) relative to methacrylated groups. Cryo-FESEM micrographs revealed that mannan-C(16) formed irregular spherical macromolecular micelles, in this work designated as nanogels, with diameters ranging between 100 and 500 nm. The influence of the polymer degree of substitution, DS(HEMA) and DS(C(16)), on the nanogel size and zeta potential was studied by DLS at different pH values and ionic strength and as a function of mannan-C(16) and urea concentrations. Under all tested conditions, the nanogel was negatively charged with a zeta potential close to zero. Mannan-C(16) with higher DS(HEMA) and DS(C(16)) values formed larger nanogels and were also less stable over a 6 month storage period and at concentrations close to the cac. When exposed to solutions of different pH and aggressive conditions of ionic strength and urea concentration, the size of mannan-C(16) varied to some extent but was always in the nanoscale range.