A lacteal is a blunt-ended, long, tube-like lymphatic vessel located in the center of each intestinal villus that provides a unique route for drainage of absorbed lipids from the small intestine. However, key regulators for maintaining lacteal integrity are poorly understood. Here I explored whether and how the gut microbiota regulates lacteal integrity. Germ depletion by antibiotic treatment triggered lacteal regression during adulthood, and delayed lacteal maturation during the postnatal period. In accordance with the functional compromise in lipid absorption, the button-like junction between lymphatic endothelial cells, which is ultrastructurally open to permit free entry of dietary lipid into lacteals, was significantly diminished in lacteals of germ-depleted mice. Lacteal defect was also found in germ-free mice, but conventionalization of germ-free mice lead to normalization of lacteals in the villi. Mechanistically, VEGF-C secreted from villi macrophages upon MyD88-dependent recognition of microbes and their products is a main factor in lacteal integrity. Collectively, I conclude that gut microbiota is a crucial regulator for lacteal integrity by endowing its unique microenvironment and regulating villi macrophages in small intestine.