Chitosan (CS) shows in vitro and in vivo efficacy for siRNA delivery but with contradictory findings for incompletely characterized systems. For understanding which parameters produce effective delivery, a library of precisely characterized chitosans was produced at different degrees of deacetylation (DDAs) and average molecular weights (M-n). Encapsulation and transfection efficiencies were characterized in vitro. Formulations were selected to examine the influence of M-n and N:P ratio on nanoparticle uptake, metabolic activity, genotoxicity, and in vitro transfection. Hemocompatibility and in vivo biodistribution were then investigated for different M-n, N:P ratios, and doses. Nanoparticle uptake and gene silencing correlated with increased surface charge, which was obtained at high DDA and high M-n. A minimum polymer length of similar to 60-70 monomers (similar to 10 kDa) was required for stability and knockdown. In vitro knockdown was equivalent to lipid control with no metabolic or genotoxicity. An inhibitory effect of serum on biological performance was dependent on DDA, M-n, and N:P. In vivo biodistribution in mice show accumulation of nanoparticles in kidney with 40-50% functional knockdown.