The Notch signaling pathway plays an important role in the bone metastasis microenvironment. Although recent evidence suggests that Notch signaling contributes to bone metastasis in breast and prostate cancer, its role and possible mechanisms in non-small cell lung cancer (NSCLC) bone metastasis are not yet clear. Here, we show that Notch3 is overexpressed in NSCLC bone metastases. The inhibition of Notch3 by small interfering RNA transfection decreased the invasion ability of NSCLC cells and transforming growth factor (TGF)-induced interleukin (IL)-6 and parathyroid hormone-related protein (pTHrP) expression in vitro. We also observed that Notch3 induced a strong morphological transformation, promoting the epithelial-mesenchymal transition (EMT). Western blotting and real-time polymerase chain reaction assays revealed that the forced overexpression of Notch3 induced the expression and activity of ZEB-1 and subsequent suppression of E-cadherin and upregulation of fibronectin, contributing to EMT and invasion. Western blotting and immunofluorescence assays showed that RNA interference-mediated ZEB-1 suppression blocked Notch-induced EMT-like transformation and subsequently reversed the observed effects on E-cadherin and downregulated fibronectin. A luciferase reporter system showed that Notch-induced ZEB-1 requires a functional binding site in the ZEB-1 promoter. In vitro invasion assays showed that the inhibition of ZEB-1 can decrease Notch3-promoted invasion and the expression of pTHrP and IL-6. Our results demonstrated that Notch upregulates ZEB-1, which contributes to TGF-β-induced EMT-like transformation and bone metastasis in NSCLC.