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Background. Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) has been shown to induce clinical response and remission in patients with active Crohn’s disease (CD). Moreover the beneficial role of GM-CSF was shown in a mouse model of acute colitis induced by Dextran Sodium Sulfate (DSS). Since it is known that GM-CSF has pleiotropic effects on monocytes and since those cells represent the exclusive source of macrophages in inflamed intestinal mucosa we characterized GM-CSF treated mouse monocytes in vitro and analyzed their function in vivo in a model of chronic DSS colitis. Methods. Mouse bone marrow-derived monocyte precursors were treated for 48 hours with GM-CSF in vitro and phenotypical changes were assessed by real-time polymerase chain reaction (qRT-PCR) and flow cytometry. Functional properties were evaluated in mixed lymphocyte reactions (MLR), by phagocytosis assays, adherence to plastic surface, cytokine production upon lipopolysaccharide (LPS) stimulation and measurement of reactive oxygen species (ROS) production. Therapeutic effects of GM-CSF-treated monocytes were evaluated in a model of chronic colitis induced by repeated oral administration of DSS (2%) on three days per week. GM-CSF treated monocytes were administered i.v. one day prior start of the third DSS treatment cycle and the severity of developed colitis was evaluated by body weight change, colon length, histology and expression of inflammatory markers. The distribution of injected GM-CSF-monocytes across intestine was evaluated by in vivo near infrared imaging. Results. We found that GM-CSF-treated monocytes express higher levels of anti-inflammatory molecules on mRNA and protein levels (e.g. IL1-Ra, IL4-R and ARG1). Functionally, GM-CSF promotes ROS production of the cells but reduces phagocytosis and adherence. In MLR we observed an induction of Foxp3 in naive T cells. Clinically, in chronic DSS-induced colitis, treatment of mice with GM-CSF-monocytes showed an overall better outcome compared to mice treated with control monocytes. This encompasses body weight, colon length, mRNA expression of inflammatory marker molecules (e.g. IL6 and TNF) and histological observations. Using in vivo imaging technology we found that a higher percentage of GM-CSF treated monocytes enter the inflamed intestine compared to control monocytes. In addition, GM-CSF-monocytes persisted longer at sides of inflammation. Conclusion. We showed that the beneficial effects of GM-CSF in CD are probably due to its modifying action especially on monocytes and concomitant anti-inflammatory alterations in phenotype and function of macrophages developed from these cells in an experimental model of colitis.