The enteric nervous system (ENS), also known as the “gut brain”, is an autonomous network of thousands of interconnected ganglia located within the walls of the digestive tract that regulates peristalsis, gut secretion and water balance. During development, the ENS is derived from “vagal” neural crest cells that emigrate from the hindbrain and, in response to environmental cues, migrate ventrally toward and caudally along the primitive gut. Once reaching their final destinations along the gut, they differentiate into distinct types of enteric neuron or glia. Whereas the migration of neural crest cells along the gut has been extensively studied, little is known about vagal neural crest migration toward the gut and the extrinsic signaling factors regulating their gut entry. Using FACS sorting, transcriptome and perturbation analyses, we have discovered that Retinoic Acid (RA) signaling pathway components are expressed in the zebrafish gut endoderm concomitant with invasion of the gut. Exogenous treatment with RA prior to and during vagal neural crest gut entry enhanced migratory progress, increased the number of gut resident neural crest cells and later of differentiated enteric neurons. Conversely, temporal attenuation of the RA pathway using Tg(hsp70:dnzRAR) heat-shocked embryos led to delayed gut entry by neural crest cells and live imaging revealed altered neural crest migratory behavior and progress along the gut. RA treatment was sufficient to expand meis3 and shha expression domains, while its attenuation led to diminished meis3 and hoxb5b expression. Together, these results suggest that the RA signaling pathway plays a key temporal role during the early phases of ENS development, thus enhancing our understanding of the genesis of the ENS in vivo.