The ability to specifically silence genes by RNA interference has an enormous potential for treating genetic diseases. However, different drawbacks such as short interfering RNA (siRNA) degradability by serum nucleases and biodistribution issues still need to be overcome to develop suitable delivery vehicles that have been proven essential in carrying siRNA to its target. Chitosan is an attractive biomaterial to construct gene nanocarriers as it is safe, cheap, and amenable to chemical modifications. However, the transfection efficiency of nanovectors based on unmodified chitosan has revealed to be relatively low and dependent on different factors such as the biopolymer molecular weight, deacetylation degree, charge ratio, pH, or particle size. Thus, specific strategies have been adopted to improve the transfection efficacy of chitosan-based nanovectors. In this work, hydrophobically modified chitosans with 8-, 10-, and 12-carbon side chains grafted to the polymeric backbone by a reductive amination process were used to develop polymeric nanoparticles by the ionotropic gelation method. After chitosan modification, the produced nanoparticles showed a suitable combination of size and surface charge with high siRNA loading capacities, efficient protection against serum nucleases, and satisfactory in vitro release profiles. Importantly, the introduced structural modifications were observed to modulate the overall physicochemical characteristics of the nanoparticles including their biological performance like their cell viability, uptake, and transfection efficiency. In this regard, the knockdown activity of the prepared nanoparticles was tested in HeLa cells overexpressing the green fluorescent protein after 24 and 48 h of incubation, observing a silencing activity greater than that displayed by the commercial transfection agent Lipofectamine 2000.