ABCB5 is a member of the ATP-binding cassette transporter superfamily that is expressed as a full transporter (ABCB5FL) and half transporter (ABCB5β). The ABCB5FL transporter mediates low-level multidrug resistance in cancer and is normally expressed in the prostate and testis, while ABCB5β has been found to be a marker of melanoma and limbal stem cells and is expressed in pigmented cells. To explore ABCB5’s role in normal physiology, we generated Abcb5-deficient C57BL/6J mice by the deletion of Abcb5 exon 2, knocking out both forms of ABCB5, which were completely phenotyped. The mice were fertile and demonstrated altered bioenergetics and fat metabolism, along with alterations in their blood composition, including anisocytosis and decreased white blood cells and platelet counts. This study uncovers further avenues of investigation into the role of Abcb5 in intermediary metabolism, particularly in relation to atherogenesis.