Background We previously reported that osteopontin plays an essential role in accelerating both reparative fibrosis and clearance of dead cells (efferocytosis) during tissue repair after myocardial infarction (MI) and galectin-3hiCD206+ macrophages is the main source of osteopontin in post-MI heart. Interleukin-10- STAT3 (signal transducer and activator of transcription 3)-galectin-3 axis is essential for Spp1 (encoding osteopontin) transcriptional activation in cardiac macrophages after MI. Here, we investigated the more detailed mechanism responsible for functional maturation of osteopontin-producing macrophages. Methods and Results In post-MI hearts, Spp1 transcriptional activation occurred almost exclusively in MerTK (Mer tyrosine kinase)+ galectin-3hi macrophages. The induction of MerTK on galectin-3hi macrophages is essential for their functional maturation including efferocytosis and Spp1 transcriptional activity. MerTK+galectin-3hi macrophages showed a strong activation of both STAT3 and ERK (extracellular signal-regulated kinase). STAT3 inhibition suppressed the differentiation of osteopontin-producing MerTK+galectin-3hi macrophages, however, STAT3 activation was insufficient at inducing Spp1 transcriptional activity. ERK inhibition suppressed Spp1 transcriptional activation without affecting MerTK or galectin-3 expression. Concomitant activation of ERK is required for transcriptional activation of Spp1. In Il-10 knockout enhanced green fluorescent protein-Spp1 knock-in mice subjected to MI, osteopontin-producing macrophages decreased but did not disappear entirely. Interleukin-10 and macrophage colony-stimulating factor synergistically activated STAT3 and ERK and promoted the differentiation of osteopontin-producing MerTK+galectin-3hi macrophages in bone marrow-derived macrophages. Coadministration of anti-interleukin-10 plus anti-macrophage colony-stimulating factor antibodies substantially reduced the number of osteopontin-producing macrophages by more than anti-interleukin-10 antibody alone in post-MI hearts. Conclusions Interleukin-10 and macrophage colony-stimulating factor act synergistically to activate STAT3 and ERK in cardiac macrophages, which in turn upregulate the expression of galectin-3 and MerTK, leading to the functional maturation of osteopontin-producing macrophages.Keywords: MerTK; galectin‐3; macrophage; macrophage colony‐stimulating factor; myocardial infarction; osteopontin.