CSF-1, by binding to its high-affinity receptor CSF-1R, sustains the survival and proliferation of monocyte/macrophages, which are central cells of innate immunity and inflammation. The MAPK ERK5 (also known as big MAPK-1, BMK1, or MAPK7) is a 98-kDa molecule sharing high homology with ERK1/2. ERK5 is activated by oxidative stress or growth factor stimulation. This study was undertaken to characterize ERK5 involvement in macrophage signaling that is elicited by CSF-1. Exposure to the CSF-1 of primary human macrophages or murine macrophage cell lines, as well as murine fibroblasts expressing ectopic CSF-1R, resulted in a rapid and sustained increase of ERK5 phosphorylation on activation-specific residues. In the BAC1. 2F5 macrophage cell line, ERK5 was also activated by another mitogen, GM-CSF, while macrophage activators such as LPS or IFN-γ and a number of nonproliferative cytokines failed. Src family kinases were found to link the activation of CSF-1R to that of ERK5, whereas protein kinase C or the serine phosphatases PP1 and PP2A seem not to be involved in the process. Treatment of macrophages with ERK5-specific small interfering RNA markedly reduced CSF-1-induced DNA synthesis and total c-Jun phosphorylation and expression, while increasing the expression of the cyclin-dependent kinase inhibitor p27. Following CSF-1 treatment, the active form of ERK5 rapidly translocated from cytosol to nucleus. Taken together, the results reported in this study show that ERK5 is indispensable for optimal CSF-1-induced proliferation and indicate a novel target for its control.