Certain oligodeoxynuclotides with CpG motifs provide enhanced immune response to co-delivered antigens. We performed a phase I, observer-blinded, randomized study in healthy anti-hepatitis B surface antigen (anti-HBsAg) antibody negative adults to explore safety and immunogenicity of co-injection of recombinant HBsAg combined with an immunostimulatory DNA sequence (ISS) 1018 ISS. Four ISS dosage groups (N=12 per group) were used: 300, 650, 1000 or 3000μg. For each group, two controls received 20μg HBsAg alone, two controls received ISS alone, and eight subjects received ISS+20μg HBsAg. Subjects received two doses 8 weeks apart. Injection site reactions (tenderness and pain on limb movement) were more frequent at higher ISS+HBsAg doses but were mainly mild and of short duration. Higher anti-HBsAg antibody levels were associated with higher ISS doses. Four weeks after the first dose, a seroprotective titer (≥10mIU/ml) was noted for 0, 25, 75, and 87.5% of subjects by increasing ISS dose group (P<0.05) for those who received ISS+HBsAg; 1 month after the second dose this increased to 62.5, 100, 100, and 100%, respectively. Geometric mean anti-HBsAg antibody levels by increasing ISS+HBsAg dose were 1.22, 5.78, 24.75, and 206.5mIU/ml after the first dose and 65.37, 877.6, 1545, and 3045mIU/ml after the second dose. We conclude that 1018 ISS+HBsAg was well tolerated and immunogenic in this phase I study in healthy adults and may offer the potential for enhancement of hepatitis B virus (HBV) immunization and protection after one or two doses or in individuals who fail to respond to the standard vaccine regimen.