Introduction: This study examined the protein and messenger RNA (mRNA) expression of molecules associated with M2 (wound healing) macrophages in mineral trioxide aggregate (MTA)-implanted rat subcutaneous tissue to elucidate the involvement of M2 macrophages in the connective tissue response to MTA. Methods: Silicone tubes containing freshly mixed MTA or a calcium hydroxide cement (Life; Kerr, Romulus, MI) were subcutaneously implanted into the backs of Wistar rats. Solid silicone rods implanted in different animals served as controls. The specimens were then double immunostained for ED1 (CD68, a general macrophage marker) and ED2 (CD163, an M2 macrophage marker). Immunostaining for CD34 (a marker for vascularization and wound healing) was also performed. Expression levels of CD34, CD163, and mannose receptor c type 1 (an M2 macrophage marker) mRNAs were determined with real-time polymerase chain reaction. Results: MTA-implanted subcutaneous tissues showed significant increases in the density of ED1+ED2+ macrophages beneath the implantation site and expression levels of CD163 and MMR mRNAs compared with Life-implanted and control tissues. MTA-implanted subcutaneous tissues also showed a significant increase of CD34-immunostained areas and up-regulation of CD34 mRNAs compared with Life-implanted and control tissues. Conclusions: MTA implantation induced the accumulation of M2 macrophage marker (ED2)-expressing macrophages and enhanced the expression of M2 macrophage marker genes. MTA implantation also enhanced the expression of CD34, suggesting acceleration of the healing/tissue repair process. Taken together, biological connective tissue response to MTA may involve wound healing/tissue repair processes involving M2 macrophages.