Short-term high fat consumption stimulates mouse islet β-cell replication through unknown mechanisms. Resident macrophages (Ms) are capable of secreting various factors involved in islet development and tissue remodeling. We hypothesized that short-term high fat diet (HFD) promotes M-infiltration in pancreatic islets and that Ms serve as regulators of β-cell replication. To test these hypotheses and dissect mechanisms involved in HFD-induced β-cell replication, adult C57BL/6J mice were fed HFD for 7 days, with or without administration of clodronate-containing liposomes, an M-depleting agent. Mouse body- and epididymal fat pad weights, and non-fasting blood glucose and fasting serum insulin levels were measured; and pancreatic islet β-cell replication, oxidative stress, apoptosis, and M infiltration were examined. Short-term HFD promoted an increase in body and epididymal fat pad weights and blood glucose levels, along with an increased fasting serum insulin concentration. β-cell replication, islet M infiltration, and the percentage of iNOS-positive Ms in the islets increased significantly in mice fed HFD. Immunofluorescence staining for 8-OHdG or activated caspase-3 revealed no significant induction of DNA damage or apoptosis, respectively. In addition, no change in stromal derived factor-1 expressing cells was found in the short-term HFD group. Despite continuous elevation of non-fasting blood glucose and fasting serum insulin levels, depletion of Ms through treatments of clodronate abrogated HFD-induced β-cell replication. These findings demonstrated that the HFD-induced M infiltration is responsible for β-cell replication. This study suggests the existence of macrophage-mediated mechanisms in β-cell replication that are independent of insulin-resistance.