Microphthalmia-associated transcription factor (MITF), a basic helix-loop-helix leucine zipper transcription factor, can govern gene expression by binding to E box elements in the promoter region of its target gene. Although high levels of MITF have been observed in cardiomyocytes and the heart, the role of MITF after myocardial infarction (MI) remains unclear. We investigated the association between substance P (SP)/neurokinin-1 receptor (NK₁R) signaling and MITF expression after MI. Male Sprague-Dawley rats (8 weeks) were randomly divided in two groups: ischemia/reperfusion injury (I/R) and SP injection (5 nmol/kg, SP+I/R). At the end of 7 days, the left ventricle (LV; LV^7daysI/R, LV^7daysSP+I/R) and infarct-related areas (IA; IA^7daysI/R, IA^7daysSP+I/R) from the hearts were collected. Immunofluorescence staining demonstrated that the LV^7daysSP+I/R had a larger population of c-Kit⁺ GATA4^high cells, which markedly upregulated MITF, c-Kit, and GATA4. c-Kit⁺ cells in the explant-derived cells (EDCs) derived from IA^7daysSP+I/R migrated more widely than EDCs IA^7daysI/R. Immunofluorescence staining, western blot analysis, and qRT-PCR assay showed that SP-treated c-Kit⁺ cells exhibited a high expression of c-Kit, GATA4, and MITF. FTY720 (a MITF inhibitor), RP67580 (NK₁R inhibitor), or both inhibited the migration and proliferation of c-Kit⁺ cells increased by SP and blocked the upregulation of c-Kit, GATA4, and MITF. Overall, we suggest that MITF might be a potential regulator in SP-mediated c-Kit⁺ cell expansion post-MI via c-Kit and GATA4.