Renal ischemia reperfusion injury (IRI) is a major factor responsible for acute renal failure (ARF). An intermediate in heme synthesis, 5-aminolevulinic acid (5-ALA), is fundamental in aerobic energy metabolism. Hemeoxygenase-1 (HO-1) cleaves heme to form biliverdin, carbon monoxide (CO) and iron (Fe²⁺), which is used with 5-ALA. In the present study, we investigated the role of 5-ALA in attenuating acute renal ischemic-reperfusion injury (IRI) using a mouse model. Male Balb/c mice received 30 mg/kg of 5-ALA with Fe²⁺ 48, 24 and 2 hours prior to IRI, and were subsequently subjected to bilateral renal pedicle occlusion for 45 minutes. The endogenous CO concentration of the kidneys from the mice administered 5-ALA/Fe²⁺ increased significantly, and the peak concentrations of serum creatinine and BUN decreased. The 5-ALA/Fe²⁺ treatments significantly decreased the tubular damage and the number of apoptotic cells. The IRI-induced renal TBARS level was also significantly decreased in the 5-ALA/Fe²⁺ group. Furthermore, the mRNA expression of HO-1, TNF-α and IFN- was significantly increased following IRI. The levels of HO-1 was increased and TNF-α and IFN- were decreased in the 5-ALA/Fe²⁺-pretreated renal parenchyma after IRI. F4/80 staining showed reduced macrophage infiltration, and TUNEL staining revealed that there were fewer interstitial apoptotic cells. These findings suggest that 5-ALA/Fe²⁺ can protect the kidneys against IRI by reducing macrophage infiltration and decreasing the renal cell apoptosis via the generation of CO.