1,25(OH)2D3 (vitamin D) appears essential for the normal development of dopaminergic neurons. Vitamin D affects dopamine synthesis and metabolism as well as expression of glial cell line–derived neurotrophic factor (GDNF), which is crucial for the survival of dopaminergic neurons. We investigated the role of vitamin D on GDNF and its receptors protooncogene tyrosine–protein kinase receptor Ret (C‐Ret) and GDNF family receptor alpha 1 (GFRα1) signaling. To this end, we used a developmental vitamin D–deficient rat model and SH‐SY5Y cells transfected with vitamin D receptor (VDR). The absence of vitamin D ligand in gestation reduces C‐Ret expression, but not GDNF and GFRα1, in embryo forebrains. Overexpression of VDR in SH‐SY5Y in the absence of ligand (mimicking in vivo developmental vitamin D deficiency) also suppressed C‐Ret mRNA levels. In the presence of vitamin D, C‐Ret mRNA and protein expression were increased. The chromatin immunoprecipitation results suggested that C‐Ret is directly regulated by vitamin D via VDR. GDNF was also increased by vitamin D in these cells. Our small interfering RNA studies showed that knocking down VDR leads to an increase in C‐Ret in the absence of ligand. Finally, we confirmed the inverse relationship between GFRα1 and C‐Ret, as knocking down C‐Ret led to increases in GFRα1 expression. These data extend our knowledge of the diverse and important roles played by vitamin D in dopamine physiology.—Pertile, R.A.N., Cui, X., Hammond, L., Eyles, D.W. Vitamin D regulation of GDNF/Ret signaling in dopaminergic neurons. FASEB J. 32, 819–828 (2018). www.fasebj.org