Duck virus hepatitis caused by duck hepatitis A virus (DHAV) is an acute and contagious disease. To better understand the pathogenic mechanism of DHAV-3 in ducklings, an infection experiment was performed. Our results showed that typical symptoms were observed in the infected ducklings. DHAV-3 could infect many tissues, leading to pathological lesions, especially on the livers and spleen, and the host immune responses are activated in infection. Real-time quantitative PCR demonstrated that expression of many innate immune-related genes was mostly up-regulated in the livers and spleen, and antiviral innate immune response was established, but not sufficient to restrict the virus replication of lethal dose. Many major pattern recognition receptors (PRRs) (RIG-1, MDA5, and TLR7) are involved in the host immune response to DHAV-3, and the expression of interferon (IFNα, IFNβ and IFNγ) and antiviral proteins (MX, OAS and PKR) are also up-regulated in the liver and spleen. The expression of most cytokines (IL-1β, IL-2 and IL-6) was also up-regulated to different degrees and was various; the expression of IL-2 increased most significantly in liver. Our data provide a foundation for further study of the pathogenicity of duck virus hepatitis and extend our understanding of the immune responses of ducklings to DHAV-3 infection.