CD4+CD25highFoxp3+ T cells suppress excess immune responses that lead to autoimmune and/or inﬂammatory diseases, and maintain host immune homeostasis. However, CD4+CD25highFoxp3+ T cells reportedly contribute to disease progression by over suppressing immune responses in some chronic infections. In this study, kinetic and functional analyses of CD4+CD25highFoxp3+ T cells were performed in cattle with bovine leukemia virus (BLV) infections, which have reported immunosuppressive characteristics. In initial experiments, production of the Th1 cytokines IFN-γ and TNF-α was reduced in BLV-infected cattle compared with uninfected cattle, and numbers of IFN-γ or TNF-α producing CD4+T cells decreased with disease progression. In contrast, IFN-γ production by NK cells was inversely correlated with BLV proviral loads in infected cattle. Additionally, during persistent lymphocytosis disease stages, NK cytotoxicity was depressed as indicated by low expression of the cytolytic protein perforin. Concomitantly, total CD4+CD25highFoxp3+ T cell numbers and percentages of TGF-β+ cells were increased, suggesting that TGF-β plays a role in the functional declines of CD4+ T cells and NK cells. In further experiments, recombinant bovine TGF-β suppressed IFN-γ and TNF-α production by CD4+ T cells and NK cytotoxicity in cultured cells. These data suggest that TGF-βfrom CD4+CD25highFoxp3+ T cells is immunosuppressive and contributes to disease progression and the development of opportunistic infections during BLV infection.