Hemoglobin (Hb) serves as the main oxygen transporter in erythrocytes, but it is also expressed in nonhematopoietic organs, where it serves an unknown function. In this study, microarray and proteomic analyses demonstrated Hb expression in the kidney. Rat kidneys were perfused extensively with saline, and glomeruli were isolated by several techniques (sieving, manual dissection, and laser capture-microdissection). Reverse transcriptase-PCR revealed glomerular alpha- and beta-globin expression, and immunoblotting demonstrated expression of the protein. In situ hybridization studies showed expression of the globin subunits in the mesangium, and immunostaining confirmed this localization of Hb. Furthermore, globin mRNA expression was detected in primary cultures of rat mesangial cells but not in cultured glomerular endothelial or epithelial cells. For investigation of Hb function in mesangial cells, the SV40-MES13 murine mesangial cell line was transfected with a vector expressing alpha- and beta-globins; this overexpression reduced production of hydrogen peroxide-induced intracellular radical oxygen species and enhanced cell viability against oxidative stress. In summary, Hb is expressed by rat mesangial cells, and its potential functions may include antioxidative defense.