The enteric nervous system arises mainly from vagal and sacral neural crest cells that colonise the gut between 9.5 and 14 days of development in mice. Using the Cre-LoxP system, we removed β1 integrins in the neural crest cells when they emerge from the neural tube. β1-null enteric neural crest cells fail to colonise the gut completely, leading to an aganglionosis of the descending colon, which resembles the human Hirschsprung's disease. Moreover,β1-null enteric neural crest cells form abnormal aggregates in the gut wall, leading to a severe alteration of the ganglia network organisation. Organotypic cultures of gut explants reveal that β1-null enteric neural crest cells show impaired adhesion on extracellular matrix and enhanced intercellular adhesion properties. They display migration defects in collagen gels and gut tissue environments. We also provide evidence that β1 integrins are required for the villi innervation in the small intestine. Our findings highlight the crucial roles played by β1 integrins at various steps of enteric nervous system development.