The hemoglobin‐vesicle (HbV) is an artificial oxygen carrier encapsulating a concentrated hemoglobin solution in a phospholipid vesicle (liposome). During or after transporting oxygen, macrophages capture HbVs in the reticuloendothelial system (RES) with an approximate circulation half‐life of 3 days. Animal studies show transient splenohepatomegaly after large doses, but HbVs were completely degraded, and the components were excreted in a few weeks. If a blood substitute is used for emergency use until red blood cell transfusion becomes available or for temporary use such as a priming fluid for an extracorporeal circuit, then one option would be to remove HbVs from the circulating blood without waiting a few weeks for removal by the RES. Using a mixture of beagle dog whole blood and HbV, we tested the separation of HbV using a centrifugal Fresenius cell separator and an ultrafiltration system. The cell separator system separated the layers of blood cell components from the HbV‐containing plasma layer by centrifugal force, and then the HbV was removed from plasma phase by the ultrafiltration system. The HbVs (250–280 nm) are larger than plasma proteins (<22 nm diameter) but smaller than blood cell components (>3 µm). The size of HbVs is advantageous to be separated from the original blood components, and the separated blood components can be returned to circulation.