Therapeutics based on small interfering RNA (siRNA) have promising potential as antiviral and anti-inflammatory agents. To deliver siRNA across cell membranes to reach the RNAi pathway in the cytosol of target cells, non-viral nanoparticulate delivery approaches are explored. Recently, we showed that encapsulation of siRNA in lipid-polymer hybrid nanoparticles (LPNs), based on poly(DL-lactic-co-glycolic acid) (PLGA) and cationic lipid-like materials (lipidoids), remarkably enhances intracellular delivery of siRNA as compared to siRNA delivery with LPNs modified with dioleoyltrimethylammoniumpropane (DOTAP) as the lipid component. However, the potential immune modulation by these cationic lipids remains unexplored. By testing lipidoids and DOTAP for innate immune-receptor-activating properties in vitro, we found that neither lipidoids nor DOTAP activate human Toll-like receptor (TLR) 2, 3, 7, and 9. However, in contrast to DOTAP, lipidoids are strong agonists for TLR4 and activate murine antigen-presenting cells in vitro. This agonistic effect was further confirmed in silico using a prediction model based on crystal structures. Also, lipidoids formulated as lipoplexes or as stable nucleic acid lipid particles, which was the reference formulation for siRNA delivery, proved to activate TLR4. However, by combining lipidoids with PLGA into LPNs, TLR4 activation was abrogated. Thus, lipidoid-mediated TLR4 activation during siRNA delivery may be modulated via optimization of the formulation design.