Dendritic cells (DC) and macrophages play important roles in maintaining the intestinal homeostasis.However, the molecular mechanisms that regulate the differentiation and responses of intestinal DC and macrophages remain poorly understood.Here, we identified microRNA (miR)-223 as a key molecule for regulating these processes.MiR-223 deficiency led to a significantly decreased number of intestinal CX3CR1hi macrophages in steady state.Both intestinal CX3CR1hi macrophages and CD103+ conventional DC (cDC) in miR-223 deficient mice exhibited a strong pro-inflammatory phenotype.Moreover, miR-223 deficient monocytes gave rise to more monocyte-derived DC (moDC) and produced more pro-inflammatory cytokines upon stimulation.Using a mouse model of colitis, we demonstrated that the miR-223 deficiency resulted in more severe colitis.Target gene analysis further demonstrated that the effects of miR-223 on DC and macrophages were mediated by directly targeting C/EBPβ.Taken together, our study identified a new role for miR-223 as a critical regulator of intestinal homeostasis.