Infectious canine hepatitis (ICH) is caused by canine adenovirus type 1 (CAV-1), which severely harms infected animals. Vaccination provides an effective approach to preventing canine infectious diseases. With the objective of exploring a new vaccination strategy that may prevent or cure ICH, we constructed a DNA vaccine, pVAX1-CpG-Loop, and evaluated its immune efficacy. We found that vaccination of BALB/c mice with the DNA vaccine alone, or priming with DNA vaccine and boosting with the Loop protein, resulted in the following: (1) High-level specific antibody (IgG) against CAV-1 was induced; (2) T cell activation was elicited; and (3) neutralizing antibodies were detectable in immunized mice. Collectively, these data indicate that the availability of a DNA vaccine could prevent hepatitis contagiosa canis.