Podocyte injury is an early feature of Fabry nephropathy, but the molecular mechanisms of podocyte injury are poorly understood. Lyso-Gb3 accumulates in serum in Fabry disease and increases extracellular matrix synthesis in podocytes. We explored the contribution of Notch1 signaling, a mediator of podocyte injury, to lyso-Gb3-elicited responses in cultured human podocytes. At clinically relevant concentrations, lyso-Gb3 activates podocyte Notch1 signaling, resulting in increased active Notch1 and HES1, a canonical Notch transcriptional target. A gamma-secretase inhibitor or specific Notch1 small interfering RNA (siRNA) inhibited HES1 upregulation in response to lyso-Gb3. Notch1 siRNA or gamma-secretase inhibition also prevented the lyso-Gb3-induced upregulation of Notch1, Notch ligand Jagged1 and chemokine (MCP1, RANTES) expression. Notch siRNA prevented the activation of nuclear factor kappa B (NF kappa B), and NF kappa B activation contributed to Notch1-mediated inflammatory responses as the NF kappa B inhibitor, parthenolide, prevented lyso-Gb3-induced chemokine upregulation. Notch1 also mediates fibrogenic responses in podocytes as Notch siRNA prevented lyso-Gb3 upregulation of fibronectin mRNA. Supporting the clinical relevance of cell culture findings, active Notch1, Jagged1 and HES1 were observed in Fabry kidney biopsies. Lyso-Gb3 elicited similar responses in mouse kidney. In conclusion, lyso-Gb3 promotes Notch1-mediated inflammatory and fibrogenic responses in podocytes that may contribute to Fabry nephropathy.