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Self-gelling DNA hydrogels with cytosine-phosphate-guanine (CpG) motifs have been shown to exhibit high potency as vaccine adjuvants. However, their oral use is limited because of their thermodynamic and chemical instability in the gastrointestinal tract. In this study, we aimed to develop DNA hydrogel microspheres (Dgel-MS) coated with chitosan to improve their stability. Chitosan-coated Dgel-MS (Cs-Dgel-MS) was prepared by emulsifying Dgel to obtain the D-gel core, followed by mixing with microemulsions of chitosan for electrostatic coating. Fluorescence imaging of Cs-Dgel-MS labeled with fluorescent dyes showed that Dgel-MS (approximately 30 μm) was coated with chitosan. The recovery efficiency of Alexa Fluor 488-DNA was 87.4 ± 7.5%. To load a phosphorothioate CpG oligodeoxynucleotide into Dgel, a modified Dgel (mDgel) was designed and fluorescein isothiocyanate (FITC)-dextran was loaded into Cs-mDgel-MS as a model compound. The recovery efficiency of Alexa Fluor 488-CpG1668 and FITC-dextran was 83.3 ± 3.8% and 67.8 ± 4.6%, respectively. The release of Alexa Fluor 488-CpG1668 from Cs-mDgel-MS was slower than that from mDgel under acidic or DNase conditions. Intra-duodenal administration of FITC-dextran/Cs-mDgel-MS showed prolonged intestinal transition of the encapsulated FITC-dextran. These results indicate that Cs-Dgel-MS can be useful for oral delivery of CpG DNA and other bioactive compounds.