The transformation of macrophages into foam cells is an important event in the development of atherosclerosis, and the oxidative injury caused by oxidized low density lipoprotein (Ox-LDL) plays an essential role in that process. It has been proved that macrophage colony-stimulating factor (M-CSF) could prevent the progression of atherosclerosis in Watanabe heritable hypercholesterolemic (WHHL) rabbits. We proposed that the anti-atherogenic effect of M-CSF was partly associated with its protective effect on monocyte-derived macrophages from Ox-LDL induced oxidative injury. In order to prove this, we investigated the effect of M-CSF on the oxidative injury caused by tert-butyl hydroperoxide (tbOOH) to mouse peritoneal macrophages and U937/J774 cell lines. The results showed that M-CSF could protect mouse peritoneal macrophages from oxidative injury (presented by cell morphology and cell survival rate); L929 cell-conditioned medium (L929-CM) had the same effect as M-CSF; and anti-M-CSF monoclonal antibody could mostly block the protective effect of L929-CM on macrophages. L929-CM was proved to be also able to decrease the impact of plasma membrane fluidity in U937 and J774 cells treated with tbOOH. Incubation with tbOOH caused DNA fragmentation in U937 cells. The presence of L929-CM greatly reduced the number of apoptotic U937 cells characterized by DNA fragmentation. From these results, we concluded that M-CSF could protect monocytes/macrophages from oxidative injury. It may be one of the mechanisms which explain the anti-atherogenic effect of exogenous M-CSF in WHHL rabbits.