Butyrophilins (BTNs) are members of the immunoglobulin superfamily commonly expressed on peripheral blood mononuclear cells (PBMCs). These molecules have a well-understood role as activators of human γδ T cells. However, several murine studies suggest that BTNs can also negatively regulate αβ T cell function. A recent study described that BTN member 3A1 (BTN3A1) overexpressed on ovarian cancer tumours attenuated CD4+ and CD8+ αβ T cell responses in humans through interaction with CD45RO, suggesting that BTN3A1 might regulate anti-cancer αβ T cell responses. To further investigate the potential role of BTN3A1 in regulating CD4+ and CD8+ αβ T cell function, we engineered BTN3A1-overexpressing K562 antigen presenting cells. Furthermore, we investigated whether BTNs interact with CD45RO by generating tetramers expressing CD45RO and BTN3A1 extracellular domain. We demonstrated that the presence of BTN3A1 did not alter αβ T cell activation, proliferation, cytokine production or K562 cell cytotoxicity. CD45RO tetramer did not bind to HEK293T cells overexpressing BTN3A1. Likewise, the BTN3A1 extracellular domain tetramer did not bind human CD45RO+ PBMCs. Hence, BTN3A1 does not appear to modulate CD4+ and CD8+ αβ T cell function, nor do they appear to interact with CD45RO which contradict published findings. Therefore, future studies are required to confirm if these BTN molecules indeed exert immunoregulatory activity on αβ T cells, and if targeting BTNs with agonist or antagonist antibodies could be beneficial for anti-cancer and viral therapeutics.