Heptahelical opioid receptors are implicated in the transcriptional regulation of neuronal development. Here we demonstrated that activation of μ-opioid receptors in human neuroblastoma SH-SY5Y cells led to the activation of signal transducer and activator of transcription 3 (STAT3), a transcription factor central to the regulation of numerous biological processes. The μ-opioid-induced activation of STAT3 is sensitive to receptor was further shown to pertussis toxin treatment and required JAK and Src tyrosine kinases, but not phosphatidylinositol 3-kinase. This μ-opioid-induced response was mediated via the extracellular signal-regulated protein kinase in a Raf-1-independent manner. The present study provides a foundation to explore the importance of STAT3 signaling in the regulation of neuronal growth and differentiation by the μ-opioid receptor.