Upregulation of muscularis macrophage numbers and activities plays an important role in the intestinal dysmotility associated with intestinal inflammation. The present study aimed to clarify changes in population dynamics of intestinal muscularis macrophages during colonic inflammation and test possible inhibitory actions of agents targeting MCP-1 on muscularis macrophage dynamics and motility disorder in the colonic inflammation elicited by 2,4,6-trinitrobenzenesulfonic acid (TNBS). In the inflamed muscle layer, ED1-positive monocytes and monocyte-derived macrophages were increased followed by increasing resident macrophages positively staining for ED2. Initiation of the ED1-positive macrophage dynamic is associated with MCP-1 mRNA expression. MCP-1 was expressed in both ED1- and ED2-positive macrophages after inflammation. Electromicroscopic analysis revealed that the cell division phase of muscularis macrophages was seen only in the early stages of inflammation. In addition, ED1 and ED2 double positive macrophages can be detected during inflammation. Treatment with dominant negative MCP-1 or neutralizing MCP-1 antibodies markedly inhibited numbers of both ED1 and ED2 positive macrophages. Inflammation-mediated dysmotility was partially recovered by treatment with neutralizing MCP-1 antibodies. These results suggest that the inflamed muscle layer is initially infiltrated by monocytes, which then differentiate and develop into muscularis-resident macrophages. These macrophages express MCP-1 for further recruitment of monocytes. MCP-1 may be one of a potential therapeutic target for inhibiting intestinal motility disorders in gut inflammation.