Spherical nucleic acids (SNAs) are a novel class of oligonucleotide agents based on densely packed oligonucleotides radially arranged on a spherical nanoparticle. As a result of their structure, SNAs exhibit increased cellular uptake and avidity for targets compared with the same oligonucleotide that is not in SNA format (linear oligo). Oligonucleotides containing CpG motifs induce TLR9-mediated immune responses. Here we designed, synthesized, and assessed immunostimulatory characteristics of TLR9 agonist SNAs using cell-based assays, and in vivo in mice and monkeys. TLR9 agonist SNAs displayed increased uptake in primary cell cultures and increased TLR9 activation in reporter cells compared with the linear oligo. In primary cell cultures TLR9 agonist SNAs induced dose-dependent TH1-type cytokine secretion and the levels of cytokines induced are relatively higher compared with the linear oligo. Subcutaneous (SC) administration of TLR9 agonist SNAs to wild-type and TLR7-/- mice induced higher levels of cytokine secretion compared with a linear oligo and no cytokine induction was observed in TLR9-/- mice, suggesting that TLR9 agonist SNAs mediate immune responses via TLR9 engagement. A single dose SC administration of TLR9 agonist SNAs to monkeys induced time- and dose-dependent TH1-type cytokines and immune cell activation. Taken together, these studies demonstrate that SNAs targeting TLR9 protein induce potent immune responses in vitro and in vivo compared with linear oligos. TLR9 agonist SNAs are a promising approach to cancer immunotherapy, asthma treatment, and vaccine adjuvant applications. One such TLR9 agonist SNA, AST-008, is being developed for immuno-oncology in combination with a selected checkpoint inhibitor.