Among new adjuvants conferring a TH1-shift, RNAdjuvant® consisting of non-coding RNA complexed with a cationic carrier peptide is a promising candidate. Upon RNAdjuvant® treatment, bone marrow-derived myeloid dendritic cells (BM-mDC) and plasmacytoid DC (BM-pDC) showed Toll-like receptor (TLR)7-dependent IFN-I induction and upregulation of CD69 and CD86. This result was verified by the ex vivo analysis of splenic DC. Immunization of mice with seasonal influenza vaccines plus RNAdjuvant® significantly enhanced vaccine-induced influenza-specific IgG responses. In particular, IgG2b and IgG2c antibody subclasses were elevated, indicating a TH1-biased immune response. RNAdjuvant® mediated enhancement of antibody responses was conferred by concomitant TLR- and RIG-I-like helicase (RLH)-dependent signaling. Thus, upon administration of RNAdjuvant® two different signaling pathways were induced, explaining the strong immunoenhancing effect of RNAdjuvant®. Notably, upon intramuscular (i.m.) injection of RNAdjuvant® IFN-I induction and CD69 upregulation on DC were only detectable in draining, but not in non-draining lymph nodes. Furthermore, transient lymphopenia as a sensitive readout of serum cytokine responses was only detected upon intravenous (i.v.), but not upon i.m., injection of RNAdjuvant®. These observations indicated that i.m. administration of RNAdjuvant® induced primarily local effects. In conclusion, RNAdjuvant® is a novel efficacious immune enhancer that augments vaccine-induced TH1 responses in a MyD88- and Cardif-dependent manner by triggering strictly local effects. Thus, RNAdjuvant® holds promise to be a particularly well tolerable new adjuvant also in humans.