In addition to conventional vaccination, DNA-mediated immunization has been developed as an alternative approach in the prevention and treatment of different infectious diseases, including hepatitis B. To define sets of serum protein and metabolite biomarkers that could be employed to determine the efficacy and safety of DNA vaccines, an integrated multiple systems biology approach was undertaken on mice immunized with DNA vaccine, recombinant protein, plasmid vector, and phosphate-buffered solution. Their sera were analyzed by two-dimensional electrophoresis and HPLC coupled with time-of-flight mass spectrometry. We detected an increase in phytosphingosine, dihydrosphingosine, palmitoylcarnitine, and ceramide in the sera of DNA-vaccinated mice. Several protein molecules were found to be altered in DNA-vaccinated mice, including apolipoprotein A-I precursor. Taken together, these results indicated that DNA vaccine stimulated hepatic sphingolipid synthesis, which may have altered the structure of circulating lipoproteins and promoted atherogenesis. This study also underscores the power of metabolomics and proteomics in the definition of DNA-vaccine-mediated metabolic phenotypes.