During development of the central and peripheral nervous systems, neurite extension mediated via glial-cell-line-derived neurotrophic factor (GDNF) and its receptor RET is critical for neuronal differentiation. In the present study, we investigated the role of the RET substrate Dok-4 in neurite outgrowth induced by the GDNF/RET signaling pathway. In TGW neuroblastoma cells, which endogenously express both RET and Dok-4, depletion of Dok-4 through treatment with small interfering RNA resulted in a marked decrease in GDNF-stimulated neurite outgrowth. By contrast, exogenous expression of wild-type Dok-4 induced sustained p44/42 mitogen-activated protein kinase (ERK1/2) activation and enhanced neurite outgrowth. Expression of Dok-4 mutants in which the tyrosine residues at codons 187, 220 and 270, conserved between Dok-4, -5, and -6, were each replaced with a phenylalanine inhibited sustained ERK1/2 activation and neurite outgrowth. We also found that Dok-4 induced a significant activation of the small G protein Rap1 and that expression of a dominant active Rap1 mutant restored neurite outgrowth in Dok-4-depleted cells. By contrast, expression of a dominant negative Rap1 mutant impaired GDNF-stimulated neurite outgrowth from TGW cells. Finally, we found that neurite formation in cultured rat hippocampal neurons was enhanced by the expression of Dok-4. Together, our results suggest that Dok-4, through activation of the Rap1-ERK1/2 pathway, regulates GDNF-mediated neurite outgrowth during neuronal development.