Updated project metadata. Resident macrophages are important for maintaining tissue homeostasis and for defence against infections, but their precise functions in different tissues are not fully elucidated.We have used high resolution quantitative proteomics to investigate the functions of splenic red pulp macrophages and peritoneal cavity macrophages in the steady-state. The validation of several proteins at cellular expression levels by the flow cytometry analysis was consistently in agreement with the proteomics data. Peritoneal macrophages were shown to be enriched in a number of key enzymes and metabolic pathways normally associated with the liver, such as metabolism of fructose, detoxification, nitrogen homeostasis and the urea cycle. Supporting observations in proteomics, we find that PM are able to utilise glutamine and glutamate which are rich in peritoneum for urea generation. In comparison, splenic red pulp macrophages were enriched in proteins important for adaptive immunity such as antigen presenting MHC molecules, in addition to proteins required for erythrocyte homeostasis and iron turnover. We also show that these tissue macrophages may utilise carbon and nitrogen substrates for different metabolic fates to support distinct tissue-specific roles. This study provides a valuable resource for biologists interested in the functions of tissue macrophages.