Clinical and experimental evidence suggests a protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx-1) in the atherogenic process. GPx-1 deficiency accelerates atherosclerosis and increases lesion cellularity in ApoE^−/− mice. However, the distribution of GPx-1 within the atherosclerotic lesion as well as the mechanisms leading to increased macrophage numbers in lesions is still unknown. Accordingly, the aims of the present study were (1) to analyze which cells express GPx-1 within atherosclerotic lesions and (2) to determine whether a lack of GPx-1 affects macrophage foam cell formation and cellular proliferation. Both in situ-hybridization and immunohistochemistry of lesions of the aortic sinus of ApoE^−/− mice after 12 weeks on a Western type diet revealed that both macrophages and – even though to a less extent – smooth muscle cells contribute to GPx-1 expression within atherosclerotic lesions. In isolated mouse peritoneal macrophages differentiated for 3 days with macrophage-colony-stimulating factor (MCSF), GPx-1 deficiency increased oxidized low density-lipoprotein (oxLDL) induced foam cell formation and led to increased proliferative activity of peritoneal macrophages. The MCSF- and oxLDL-induced proliferation of peritoneal macrophages from GPx-1^−/−ApoE^−/− mice was mediated by the p44/42 MAPK (p44/42 mitogen-activated protein kinase), namely ERK1/2 (extracellular-signal regulated kinase 1/2), signaling pathway as demonstrated by ERK1/2 signaling pathways inhibitors, Western blots on cell lysates with primary antibodies against total and phosphorylated ERK1/2, MEK1/2 (mitogen-activated protein kinase kinase 1/2), p90RSK (p90 ribosomal s6 kinase), p38 MAPK and SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase), and immunohistochemistry of mice atherosclerotic lesions with antibodies against phosphorylated ERK1/2, MEK1/2 and p90RSK. Representative effects of GPx-1 deficiency on both macrophage proliferation and MAPK phosphorylation could be abolished by the GPx mimic ebselen. The present study demonstrates that GPx-1 deficiency has a significant impact on macrophage foam cell formation and proliferation via the p44/42 MAPK (ERK1/2) pathway encouraging further studies on new therapeutic strategies against atherosclerosis.