OBJECTIVE Mesenchymal stem cells (MSCs) have been shown to produce adenosine, express adenosine receptors, and communicate with macrophages and other cells. However, there is no information about the role of caffeine, as a popular drink and adenosine antagonist, on the crosstalk between MSCs and immune cells. The aim of the current study is to evaluate the effects of the conditioned medium of MSCs treated with caffeine on macrophages. METHODS In this experimental study, MSCs were isolated from bone marrow of rats and pulsed with different concentrations of caffeine (0, 0.1, 0.5 and 1 mM) for 72 hours. The conditioned medium of MSCs was collected after 24 hours, then incubated with macrophages for 24 hours. Finally, the functions of the macrophages were evaluated. RESULTS Conditioned medium of MSCs treated with caffeine significantly enhanced phagocytosis and simultaneously regressed expression of reactive oxygen species (ROS) and nitric oxide (NO) as well as IL-12 by macrophages compared to the supernatants of MSCs alone. The conditioned medium of MSCs pulsed with caffeine at low to moderate concentrations preserved the neutral red uptake by macrophages and elevated IL-10 secretion by macrophages. A high concentration of caffeine could interfere with the two latter effects of supernatants of MSCs on the macrophages. CONCLUSIONS Collectively, caffeine treatment of MSCs appeared to augment the instruction of anti-inflammatory macrophages by conditioned medium of MSCs. These findings might offer new insight into the potential mechanisms that underlie the immunomodulatory and anti-inflammatory effects of caffeine.