The CD8αβ coreceptor is crucial for effective peptide: MHC-I recognition by the TCR of CD8+ T cells. Adenosine diphosphate ribosyl transferase 2.2 (ART2.2) utilizes extracellular NAD+ to transfer ADP-ribose to arginine residues of extracellular domains of surface proteins. Here, we show that in the presence of extracellular NAD+, ART2.2 caused ADP-ribosylation of CD8-β on murine CD8+ T cells in vitro and in vivo. Treatment with NAD+ prevented binding of anti-CD8-β mAb YTS156.7.7 but not of mAb H35–17.2, indicating that NAD+ caused modification of certain epitopes and not a general loss of CD8-β. Loss of antibody binding was strictly dependent on ART2.2, because it was not observed on ART2-deficient T cells or in the presence of inhibitory anti-ART2.2 single-domain antibodies. ADP-ribosylation of CD8-β occurred during cell isolation, particularly when cells were isolated from CD38-deficient mice. Incubation of ART2-expressing, but not of ART2-deficient, OVA-specific CD8+ T cells with NAD+ interfered with binding of OVA257–264:MHC-I tetramers. In line with this result, treatment of WT mice with NAD+ resulted in reduced CD8+ T-cell mediated cytotoxicity in vivo. We propose that ADP-ribosylation of CD8-β can regulate the coreceptor function of CD8 in the presence of elevated levels of extracellular NAD+.