Myocardial neovascularization play key role in the structural remodeling that occurs in heart failure. Prior studies have suggested an important role for splenic monocytes in tissue healing after myocardial infarction; however, the role of macrophages in regulating angiogenesis at the site of tissue injury is complex and not well defined. Moreover, it is unclear how the spleen regulates macrophage-mediated neovascularization. We hypothesized that the spleen plays a critical role in determining macrophage polarity that in turn influences tissue neovascularization. In 7 naïve and 7 splenectomized C57BL/6 mice (splenectomy performed 28 d prior), we performed in vivo Matrigel plug assays. Matrigel (0.6 mL/plug) was injected subcutaneously, either alone or together with peritoneal macrophages (∼20,000 cells/plug) isolated from syngeneic mice, and Matrigel plug neovascularization was assessed after 10 d. Masson trichrome staining and CD31 immunostaining was used to index vascularization. Circulating activated monocytes (CD11b+F480+) and endothelial progenitor cells (EPCs, CD34+Flk-1+) were assessed by flow cytometry. Flow cytometry of peripheral blood revealed significant reductions in circulating activated monocytes (2.20 ± 0.32 vs. 3.95 ± 0.49 %; p=0.002) and EPCs (0.0076 ± 0.0013 vs. 0.018 ± 0.0023; p=0.0002) in splenectomized mice as compared with naïve mice. Splenectomized mice also exhibited markedly reduced neovessel formation in Matrigel plugs indexed by morphometric and immunohistological analyses. The addition of peritoneal macrophages to the Matrigel plug restored vessel formation in splenectomized mice, but did not change neovascularization in naïve mice. Moreover, this restoration of neovascularization occurred with the addition of peritoneal macrophages polarized to an M2 phenotype (using interleukin [IL]-4 or IL-10) but not to an M1 phenotype. We conclude that the spleen is a key determinant of macrophage polarization and function that subsequently impacts the efficiency of tissue neovascularization. Hence, the spleen provides an anti-inflammatory and pro-angiogenic regulatory function in vivo that may be of central importance to the adequacy of myocardial repair and remodeling following tissue injury.