Arginase activity is expressed by macrophages in healing wounds and other sites of inflammation and has been shown to modulate the synthesis of nitric oxide, polyamines, and collagen. The role of CCAAT/enhancer-binding protein β (C/EBPβ) in the regulation of macrophage arginase by different agonists was investigated using C/EBPβ−/− and+/+ macrophage cell lines. 8-Bromo-cyclic adenosine monophosphate (8-Br-cAMP, 0.5 mM), recombinant murine interleukin 4 (rmIL-4, 20 U/mL), Escherichia coli lipopolysaccharide (100 ng/mL), and hypoxia (1% O 2) induced arginase activity in C/EBPβ+/+ macrophages, where enzyme activity correlated with arginase I protein. Only rmIL-4 increased arginase activity in C/EBPβ−/− cells. Arginase II protein was expressed constitutively in wild-type and C/EBPβ−/− cell lines and was unaltered by 8-Br-cAMP or rmIL-4. rmIL-4-stimulated immortalized C/EBPβ−/− macrophages demonstrated higher nuclear signal transducer and activator of transcription-6 (STAT6) and phospho-STAT6 content than their+/+ counterparts. Validating the biological relevance of findings with the cell lines, additional experiments examined wound fluids and peritoneal macrophages from C/EBPβ−/− mice and demonstrated that both contained less arginase activity than those from wild-type controls. Wounds in C/EBPβ−/− animals showed signs of delayed maturation, as manifested by the persistence of neutrophils in the inflammatory infiltrate. Peritoneal macrophages from C/EBPβ+/+ animals responded to 8-Br-cAMP and rmIL-4 with increased arginase activity, whereas those from C/EBPβ−/− mice did not respond to cAMP. Results demonstrate a key mechanistic role for C/EBPβ in the modulation of macrophage arginase I expression in vivo and in vitro.