Phenotypic changes of myeloid cells are critical to the regulation of premature aging, development of cancer, and responses to infection. Heme metabolism has a fundamental role in the regulation of myeloid cell function and activity. Here, we show that deletion of heme oxygenase-1 (HO-1), an enzyme that removes heme, results in an impaired DNA damage response (DDR), reduced cell proliferation, and increased cellular senescence. We detected increased levels of p16INK4a, H2AXγ, and senescence-associated-β-galactosidase (SA-β-Gal) in cells and tissues isolated from HO-1-deficient mice. Importantly, deficiency of HO-1 in residential macrophages in chimeric mice results in elevated DNA damage and senescence upon radiation-induced injury. Mechanistically, we found that mammalian target of rapamycin (mTOR)/S6 protein signaling is critical for heme and HO-1-regulated phenotype of macrophages. Collectively, our data indicate that HO-1, by detoxifying heme, blocks p16INK4a expression in macrophages, preventing DNA damage and cellular senescence.