Recent reports have shown that immunostimulatory sequences (ISS) containing CpG motifs have minimal length requirements (≥12 bases) for the exertion of immune‐enhancing function upon mammalian cells. Herein we demonstrate that short ISS (5–7 bases), which exhibit no activity on their own, induce IFN‐γ and IFN‐α secretion from human peripheral blood mononuclear cells when adsorbed to the surface of cationic poly(D,L‐lactide‐co‐glycolide) microparticles (cPLGA). Utilizing this technique, we discovered a minimal ISS sequence for induction of IFN‐γ and IFN‐α from human cells: 5′‐TCGXX‐3′. These short ISS/cPLGA formulations targeted PDC in similar fashion to longer ISS ODN, the activity of which does not require (but is enhanced by) cPLGA. PDC stimulated with short ISS/cPLGA responded with enhanced uptake of ISS and elevated production of cytokines, including IFN‐α. However, ISS‐responsive B cells did not respond to short ISS/cPLGA, underlining the plasmacytoid dendritic cell selectivity of this formulation. These results describe a novel technique for formulating active, but very short, ISS oligodeoxynucleotide that allows for the dissection and characterization of minimal immunostimulatory CpG motifs.