We evaluated the contribution of CD8αβ+ T cells to control of live-attenuated simian immunodeficiency virus (LASIV) replication during chronic infection and subsequent protection from pathogenic SIV challenge. Unlike previous reports with a CD8α-specific depleting monoclonal antibody (mAb), the CD8β-specific mAb CD8β255R1 selectively depleted CD8αβ+ T cells without also depleting non-CD8+ T cell populations that express CD8α, such as natural killer (NK) cells and γδ T cells. Following infusion with CD8β255R1, plasma viremia transiently increased coincident with declining peripheral CD8αβ+ T cells. Interestingly, plasma viremia returned to predepletion levels even when peripheral CD8αβ+ T cells did not. Although depletion of CD8αβ+ T cells in the lymph node (LN) was incomplete, frequencies of these cells were 3-fold lower (P = 0.006) in animals that received CD8β255R1 than in those that received control IgG. It is possible that these residual SIV-specific CD8αβ+ T cells may have contributed to suppression of viremia during chronic infection. We also determined whether infusion of CD8β255R1 in the LASIV-vaccinated animals increased their susceptibility to infection following intravenous challenge with pathogenic SIVmac239. We found that 7/8 animals infused with CD8β255R1, and 3/4 animals infused with the control IgG, were resistant to SIVmac239 infection. These results suggest that infusion with CD8β255R1 did not eliminate the protection afforded to LASIV vaccination. This provides a comprehensive description of the impact of CD8β255R1 infusion on the immunological composition in cynomolgus macaques, compared to an isotype-matched control IgG, while showing that the control of LASIV viremia and protection from challenge can occur even after CD8β255R1 administration.IMPORTANCE Studies of SIV-infected macaques that deplete CD8+ T cells in vivo with monoclonal antibodies have provided compelling evidence for their direct antiviral role. These studies utilized CD8α-specific mAbs that target both the major (CD8αβ+) and minor (CD8αα+) populations of CD8+ T cells but additionally deplete non-CD8+ T cell populations that express CD8α, such as NK cells and γδ T cells. In the current study, we administered the CD8β-specific depleting mAb CD8β255R1 to cynomolgus macaques chronically infected with a LASIV to selectively deplete CD8αβ+ T cells without removing CD8αα+ lymphocytes. We evaluated the impact on control of virus replication and protection from pathogenic SIVmac239 challenge. These results underscore the utility of CD8β255R1 for studying the direct contribution of CD8αβ+ T cells in various disease states.