Glial cell-line derived neurotrophic factor (GDNF) and neurturin (NTN) belong to a structurally related family of neurotrophic factors. NTN exerts its effect through a multi-component receptor system consisting of the GDNF family receptor alpha 2 (GFRα2), proto-oncogene RET and/or NCAM. GFRα2 is alternatively spliced into 3 isoforms, namely GFRα2a, GFR&agr2b, and GFRα2c. The present study focused on the expressions and functions of GFRα2 isoforms. These receptor isoforms were differentially expressed in human brain regions. Using Neuro2A model, GDNF and NTN promote neurites outgrowth by activating GFα2a and GFRα2c, but not GFRα2b. These GFRα2 isoforms regulates different early response genes when stimulated with GDNF and NTN. Interestingly, using co-expression models, GFRα2b inhibits ligand induced neurites outgrowth of GFRα2a and GFRα2c, and also the related receptor, GFRα1a. More intriguingly, ligands activated GFRα2b was also able to attenuate neuritogenesis induced by retinoic acid. We further investigated the mechanism involved in GFRα2b antagonistic activity. We found that MAPK induced by GDNF was not attenuated by GFRα2b in a co-expression model, while early response genes regulation were dominated by GFRα2b, which up-regulated FosB. These suggest that GFRα2b is not merely a dominant negative isoforms, but it utilizes a yet to be characterized mechanism in antagonism and inhibition of neuritogenesis. Together, these data suggest a novel paradigm for the regulation of growth factor signaling and neurites outgrowth via an inhibitory splice variant of the receptor.