Neurite outgrowth is an essential process during neuronal differentiation as well as neuroregeneration. Thus, understanding the molecular and cellular control of neurite outgrowth will benefit patients with neurological diseases. We have previously shown that overexpression of the signaling adaptor protein SH2B1β promotes fibroblast growth factor 1 (FGF1)-induced neurite outgrowth (W. F. Lin, C. J. Chen, Y. J. Chang, S. L. Chen, I. M. Chiu, and L. Chen, Cell. Signal. 21:1060-1072, 2009). SH2B1β also undergoes nucleocytoplasmic shuttling and regulates a subset of neurotrophin-induced genes. Although these findings suggest that SH2B1β regulates gene expression, the nuclear role of SH2B1β was not known. In this study, we show that SH2B1β interacts with the transcription factor, signal transducer, and activator of transcription 3 (STAT3) in neuronal PC12 cells, cortical neurons, and COS7 fibroblasts. By affecting the subcellular distribution of STAT3, SH2B1β increased serine phosphorylation and the concomitant transcriptional activity of STAT3. As a result, overexpressing SH2B1β enhanced FGF1-induced expression of STAT3 target genes Egr1 and Cdh2. Chromatin immunoprecipitation assays further reveal that, in response to FGF1, overexpression of SH2B1β promotes the in vivo occupancy of STAT3-Sp1 heterodimers at the promoter of Egr1 and Cdh2. These findings establish a central role of SH2B1β in orchestrating signaling events to transcriptional activation through interacting and regulating STAT3-containing complexes during neuronal differentiation.