Nucleic acid nanoparticles (NANPs) have evolved as a new class of therapeutics with the potential to detect and treat diseases. Despite tremendous advancements in NANP development, their immunotoxicity, one of the major impediments in clinical translation of traditional therapeutic nucleic acids (TNAs), has never been fully characterized. Here, we describe the first systematically studied immunological recognition of 25 representative RNA and DNA NANPs selected to have different design principles and physicochemical properties. We discover that, unlike traditional TNAs, NANPs used without a delivery carrier are immunoquiescent. We show that interferons (IFNs) are the key cytokines triggered by NANPs after their internalization by phagocytic cells, which agrees with predictions based on the experiences with TNAs. However, in addition to type I IFNs, type III IFNs also serve as reliable biomarkers of NANPs, which is usually not characteristic of TNAs. We show that overall immunostimulation relies on NANP shapes, connectivities, and compositions. We demonstrate that, like with traditional TNAs, plasmacytoid dendritic cells serve as the primary interferon producers among all peripheral blood mononuclear cells treated with NANPs, and scavenger receptor-mediated uptake and endosomal Toll-like receptor signaling are essential for NANP immunorecognition. The TLR involvement, however, is different from that expected for traditional TNA recognition. Based on these results, we suggest that NANP technology may serve as a prototype of auxiliary molecular language for communication with the immune system and the modulation of immune responses.