With the development of tissue engineering and regenerative medicine, prevascularized marrow mesenchymal stem cell (BMSC) sheets have been regarded as a promising method for tissue regeneration. Furthermore, the inflammatory response is one of the main regulators of vascularization and the restoration of engineered tissue function; among them, macrophages and cytokines produced by them are considered to be the decisive factors of the downstream outcomes. This study investigated the effect of macrophages on the formation of microvascular-like structures of human umbilical vein endothelial cells (HUVECs) in BMSC sheets. First, a human monocytic leukaemia cell line (THP-1 cells) was differentiated into derived macrophages (M0) with phorbol 12-myristate 13-acetate (PMA) and further activated into proinflammatory macrophages (M1 macrophages) with IFN-γ and LPS or anti-inflammatory macrophages (M2 macrophages) with IL-4. Then, HUVECs and prevascularized sheets were treated with conditioned media (CM) from different macrophages, and the impact of macrophage phenotypes on vascularized network formation in prevascularized cell sheets was examined by HE staining, CD31 immunofluorescence staining and ELISA. Our study showed that macrophages may guide the arrangement of endothelial cells through a paracrine pathway. Cell sheets that were cultured in the conditioned media from M2 macrophages were thinner than those cultured in other media. At various time points, the levels of TNF-α and VEGF in prevascularized sheets cultured with CM(M1) was higher than that in sheets cultured with other media; however, the levels of PDGF-BB in prevascularized sheets cultured with CM(M2) was higher than that in sheets cultured with other media. These findings suggest that the paracrine effect of macrophages can influence the formation of microvascular networks in prevascularized sheets by regulating the arrangement of cells, the thickness of the cell sheet and the secretion of cytokines related to angiogenesis. Macrophages with different phenotypes have unique effects on prevascularized sheets.