Inflammatory bowel disease (IBD) is a chronic intestinal inflammation, but the accurate etiology remains to be elucidated. Increasing evidence has shown that macrophages polarize to different phenotypes depending on the intestinal microenvironment and are associated with the progression of IBD. In the present study, we investigated the effect of oxytocin, a neuroendocrinal, and pro-health peptide, on the modulation of macrophages polarization and the progression of experimental colitis. Our data demonstrated that oxytocin decreased the sensitivity of macrophages to lipopolysaccharide stimulation with lower expression of inflammatory cytokines, like IL-1β, IL-6, and TNF-α, but increased the sensitivity to IL-4 stimulation with enhanced expression of M2-type genes, arginase I (Arg1), CD206, and chitinase-like 3 (Chil3). This bidirectional modulation was partly due to the up-regulation of β-arrestin2 and resulted in the inhibition of NF-κB signaling and reinforcement of Signal transducer and activator of transcription (STAT) 6 phosphorylation. Moreover, oxytocin receptor (OXTR) myeloid deficiency mice were more susceptible to dextran sulfate sodium (DSS) intervention compared with the wild mice. For the first time, we reveal that oxytocin–oxytocin receptor system participates in modulating the polarization of macrophages to an anti-inflammatory phenotype and alleviates experimental colitis. These findings provide new potential insights into the pathogenesis and therapy of IBD.