The gastrointestinal tract is innervated by an intrinsic neuronal network, known as the enteric nervous system (ENS), and by extrinsic axons arising from peripheral ganglia. The nerve of Remak (NoR) is an avian-specific sacral neural crest-derived ganglionated structure that extends from the cloaca to the proximal midgut and, similar to the pelvic plexus, provides extrinsic innervation to the distal intestine. The molecular mechanisms controlling extrinsic nerve fiber growth into the gut is unknown. In vertebrates, CXCR4, a cell-surface receptor for the CXCL12 chemokine, regulates migration of neural crest cells and axon pathfinding. We have employed chimeric tissue recombinations and organ culture assays to study the role of CXCR4 and CXCL12 molecules in the development of colorectal innervation. CXCR4 is specifically expressed in nerve fibers arising from the NoR and pelvic plexus, while CXCL12 is localized to the hindgut mesenchyme and enteric ganglia. Overexpression of CXCL12 results in significantly enhanced axonal projections to the gut from the NoR, while CXCR4 inhibition disrupts nerve fiber extension, supporting a previously unreported role for CXCR4 and CXCL12 signaling in extrinsic innervation of the colorectum.