The method of delivering a DNA vaccine can influence the type of immune response induced by the vaccine. Application of a DNA vaccine by gene gun typically induces a T helper type 2 (Th2)-type reaction, whereas needle inoculation triggers a Th1 response. In the present study, we found that physical trauma, gold-particle bombardment and the CpG motif can act as 'danger signals' that recruit inflammatory cells to damaged tissues. Analysis of the cytokine profiles of draining lymph nodes or lymph-node-derived mononuclear cells from different groups by real-time reverse transcription-polymerase chain reaction revealed that, while gene-gun-bombardment induced a Th2-type cytokine microenvironment with increased interleukin-4 (IL-4) and IL-10 mRNA expression and almost no increase in IL-12 and interferon-gamma mRNA expression in draining lymph nodes, intradermal injection as well as subcutaneous injection of muscle induced the opposite. We further studied whether the addition of the CpG motif can switch the Th2-type cytokine microenvironment produced by gene-gun bombardment in draining lymph nodes. Results showed that the addition of the CpG motif can increase IL-12 mRNA expression in draining lymph nodes whether induced by intradermal injection, intramuscular injection, or gene-gun bombardment. These data suggest that delivery of the CpG motif induced a Th1-biased microenvironment in draining lymph nodes. Taken together, the CpG motif can act as a 'danger signal' and Th1 immune response enhancer in DNA vaccination. These results may help to explain the mechanism of different types of immune response induced by DNA vaccines delivered by different routes and facilitate the application of DNA vaccines.