Arginine metabolism plays an important role in the activation of phagocytes, which are responsible for initiation, regulation, and resolution of inflammation. Deterioration of phagocyte functions is thought to cause the development of inflammaging and other age-related disorders. Mesenchymal stromal cells are well known for their immunoregulatory properties and ability to modulate activation status of mononuclear phagocytes. We aimed to compare arginase activity and NO production in phagocytes from different sources in young and aged animals, and to explore the effect of syngeneic thymic mesenchymal stromal cells on these parameters in tissue-resident phagocytes. We found that arginine metabolism of tissue-resident mononuclear phagocytes from aged mice is shifted to alternative or anti-inflammatory phenotype, due to a statistically significant arginase activity increase. Syngeneic co-culture with mesenchymal stromal cells greatly stimulates arginase activity and up-regulates nitric oxide generation by tissue mononuclear phagocytes regardless of cell donor age. Such bi-directional influence may be beneficial in clinical application for simultaneous inhibition of inflammation and infectious process.