Lysosomal TLR-9 is stimulated in A549 lung epithelial cells through administration of nanoparticles (NPs) either based on γ-Fe2O3 or MnO. Synthetic single-stranded immunostimulatory CpG-oligodeoxynucleotides (CpG-ODN) are covalently attached to fluorescently labelled γ-Fe2O3- and MnO-NPs in order to monitor the impact of TLR-9 activation on motility and cell morphology employing time-resolved impedance spectroscopy. In contrast to cytotoxic MnO-based particles, particles made from Fe2O3 are non-toxic carriers for pathogen-mimicking CpG-ODNs, which efficiently stimulate endogenous TLR-9, resulting in enhanced micromotility and a loss of barrier properties. Compared to neat CpG-ODNs administered in the absence of particles, the nucleotides displayed by NPs are found to be considerably more efficient in stimulating A549 cells attributed to a larger local concentration of ligands on the particles’ surface. The study shows that particle-based CpG-ODNs added to tumour cells increase their motility even further and therefore might also enhance their invasiveness and metastatic potential, foiling the original strategy of immunotherapy.