Liver resection is a common treatment for various conditions and often requires blood transfusions to compensate for operative blood loss. As partial hepatectomy (PHx) is frequently performed in patients with a pre-damaged liver, avoiding further injury is of paramount clinical importance. Our aim was to study the impact of red blood cell (RBC) resuscitation on liver regeneration. We assessed the impact of RBC storage time on liver regeneration following 50% PHx in rats and explored possible contributing molecular mechanisms using immunohistochemistry, RNA-Seq, and macrophage depletion. The liver was successfully regenerated after PHx when rats were transfused with fresh RBCs (F-RBCs). However, in rats resuscitated with stored RBCs (S-RBCs), the regeneration process was disrupted, as detected by delayed hepatocyte proliferation and lack of hypertrophy. The delayed regeneration was associated with elevated numbers of hemorrhage-activated liver macrophages (Mhem) secreting HO-1. Depletion of macrophages prior to PHx and transfusion improved the regeneration process. Gene expression profiling revealed alterations in numerous genes belonging to critical pathways, including cell cycle and DNA replication, and genes associated with immune cell activation, such as chemokine signaling and platelet activation and adhesion. Our results implicate activated macrophages in delayed liver regeneration following S-RBC transfusion via HO-1 and PAI-1 overexpression.