RET receptor signalling is essential for glial-cell-line-derived neurotrophic factor (GDNF)-induced survival and differentiation of various neurons such as mesencephalic neurons. To identify proteins that mediate RET-dependent signaling, yeast two-hybrid screening was performed with the intracellular domain of RET as bait. We identified a new interaction between RET and the adapter protein SH2-Bβ. Upon GDNF stimulation of PC12-GFRα1-RET cells (that stably overexpress GDNF receptor α1 and RET), wild-type SH2-Bβ co-immunoprecipitated with RET, whereas the dominant-negative SH2-Bβ mutant R555E did not. RET interacted with endogenous SH2-Bβ both in PC12-GFRα1-RET cells and in rat tissues. Mutagenesis analysis revealed that Tyr981 within the intracellular domain of RET was crucial for the interaction with SH2-Bβ. Morphological evidence showed that SH2-Bβ and RET colocalized in mesencephalic neurons. Furthermore, functional analysis indicated that overexpression of SH2-Bβ facilitated GDNF-induced neurite outgrowth in both PC12-GFRα1-RET cells and cultured mesencephalic neurons, whereas the mutant R555E inhibited the effect. Moreover, inhibition of SH2-Bβ expression by RNA interference caused a significant decrease of GDNF-induced neuronal differentiation in PC12-GFRα1-RET cells. Taken together, our results suggest that SH2-Bβ is a new signaling molecule involved in GDNF-induced neurite outgrowth.