Domestic ducks are the important host for H5N1 highly pathogenic avian influenza virus (HPAIV) infection and epidemiology, but little is known about the duck T cell response to H5N1 AIV infection. In infection experiments of mallard ducks, we detected significantly increased CD8(+) cells and augmented expression of cytotoxicity-associated genes, including granzyme A and IFN-gamma, in PBMCs from 5 to 9 d postinfection when the virus shedding was clearly decreased, which suggested the importance of the duck cytotoxic T cell response in eliminating H5N1 infection in vivo. Intriguingly, we found that a CD8(high+) population of PBMCs was clearly upregulated in infected ducks from 7 to 9 d postinfection compared with uninfected ducks. Next, we used Smart-Seq2 technology to investigate the heterogeneity and transcriptional differences of the duck CD8(+) cells. Thus, CD8(high+) cells were likely to be more responsive to H5N1 AIV infection, based on the high level of expression of genes involved in T cell responses, activation, and proliferation, including MALT1, ITK, LCK, CD3E, CD247, CFLAR, IL-18R1, and IL-18RAP. More importantly, we have also successfully cultured H5N1 AIV-specific duck T cells in vitro, to our knowledge, for the first time, and demonstrated that the CD8(high+) population was increased with the duck T cell activation and response in vitro, which was consistent with results in vivo. Thus, the duck CD8(high+) cells represent a potentially effective immune response to H5N1 AIV infection in vivo and in vitro. These findings provide novel insights and direction for developing effective H5N1 AIV vaccines.