Hydropericardium-hepatitis syndrome, a recently emerged disease of chickens, is caused by some strains of fowl adenovirus serotype 4 (FAdV-4). However, the relationship between the immune response and cytokine expression during FAdV-4 infection is largely unknown. In this study, our data showed that all chickens exhibited typical clinical signs and lesions and that the viral load was significantly increased in both the liver and thymus following FAdV-4 infection. We also found that the appearance of tissue lesions in the liver and thymus was consistent with the viral copy numbers, indicating that virus replication in systemic organs closely correlated with disease progression. In addition, the effects of FAdV-4 infection on the transcription of some avian cytokines were studied in vivo. In general, expression of the proinflammatory cytokines interleukin (IL)-2 and interferon (IFN)-α and IFN-β in the liver and thymus was strongly upregulated. Interestingly, the expression of IL-2 was the most highly upregulated. Expression of the anti-inflammatory cytokines IL-4, IL-10, and transforming growth factor (TGF)-β1 and TGF-β2, were also upregulated. Moreover, we investigated both the humoral and cellular immune responses in chickens infected with FAdV-4. Compared to those in the noninfected chickens, the antibody levels in chickens infected with FAdV-4 were significantly increased within 30 days postinfection. In addition, the ratio of CD4+/CD8+ T cells was decreased in FAdV-4-infected chickens. Taken together, these findings increase our understanding of the pathogenesis of FAdV-4 in chickens and provide a foundation for additional pathogenesis studies.