Abstract The administration of granulocyte colony-stimulating factor (G-CSF) after myocardial infarction (MI) improves cardiac function and survival rates in mice. It was also reported recently that bone marrow (BM)-derived c-kit+ cells or macrophages in the infarcted heart are associated with improvement of cardiac remodeling and function. These observations prompted us to examine whether BM-derived hematopoietic cells mobilized by G-CSF administration after MI play a beneficial role in the infarct region. A single hematopoietic stem cell from green fluorescent protein (GFP)-transgenic mice was used to reconstitute hematopoiesis in each experimental mouse. MI was then induced, and the mice received G-CSF for 10 days. In the acute phase, a number of GFP+ cells showing the elongated morphology were found in the infarcted area. Most of these cells were positive for vimentin and α-smooth muscle actin but negative for CD45, indicating that they were myofibroblasts. The number of these cells was markedly enhanced by G-CSF administration, and the enhanced myofibroblast-rich repair was considered to lead to improvements of cardiac remodeling, function, and survival rate. Next, G-CSF-mobilized monocytes were harvested from the peripheral blood of GFP-transgenic mice and injected intravenously into the infarcted mice. Following this procedure, GFP+ myofibroblasts were observed in the infarcted myocardium. These results indicate that cardiac myofibroblasts are hematopoietic in origin and could arise from monocytes/macrophages. MI leads to the recruitment of monocytes, which differentiate into myofibroblasts in the infarct region. Administration of G-CSF promotes this recruitment and enhances cardiac protection. Disclosure of potential conflicts of interest is found at the end of this article.