Intestinal macrophages are critical for gastrointestinal (GI) homeostasis, but our understanding of their role in regulating intestinal motility is incomplete. Here, we report that CX3C chemokine receptor 1-expressing muscularis macrophages (MMs) were required to maintain normal GI motility. MMs expressed the transient receptor potential vanilloid 4 (TRPV4) channel, which senses thermal, mechanical, and chemical cues. Selective pharmacologic inhibition of TRPV4 or conditional deletion of TRPV4 from macrophages decreased intestinal motility and was sufficient to reverse the GI hypermotility that is associated with chemotherapy treatment. Mechanistically, stimulation of MMs via TRPV4 promoted the release of prostaglandin E2 and elicited colon contraction in a paracrine manner via prostaglandin E receptor signaling in intestinal smooth muscle cells without input from the enteric nervous system. Collectively, our data identify TRPV4-expressing MMs as an essential component required for maintaining normal GI motility and provide potential drug targets for GI motility disorders. Display omitted •Macrophage-specific Trpv4-deficient mice display reduced gastrointestinal motility•Direct interactions between MMs and smooth muscle cells produce colon contraction•Enteric nervous system is not involved in macrophage-mediated colon contraction•TRPV4 inhibition reverses GI hypermotility associated with chemotherapy treatment How intestinal macrophages regulate intestinal motility remains poorly understood. Luo et al. demonstrate that muscularis macrophages expressing the TRPV4 channel promote GI motility by directly affecting the function of intestinal smooth muscle cells independent of the enteric nervous system.