The use of cyclosporine has been restricted by its nephrotoxic effects mediated, in part, by reactive oxygen species (ROS). Phosphoinositide 3-kinase, protein kinase B, and extracellular regulated kinase (ERK) pathways are related to survival and cell death and are activated after ROS generation. In this study, we evaluated the effects of cyclosporine on these pathways and their contribution to cyclosporine-induced toxicity. Viability of cells derived from the proximal tubule of transgenic mice was measured with Trypan Blue, ROS generation by a fluorescent probe, while ERK and phosphoinositide 3-kinase/protein kinase B activation were monitored with phospho-specific antibodies. Cyclosporine decreased cell viability and induced ROS generation and ERK and phosphoinositide 3-kinase activation. Both pathways were activated by the epidermal growth factor receptor (EGFR). Antioxidants blocked ERK activation but failed to inhibit protein kinase B phosphorylation or prevent cyclosporine toxicity. ERK inhibition did not protect from cyclosporine-induced cell death. EGFR or phosphoinositide 3-kinase inhibitors protected from cyclosporine-triggered cell death without decreasing ROS. Small interfering RNA against the catalytic subunit of phosphoinositide 3-kinase decreased protein kinase B phosphorylation but did not prevent cyclosporine-mediated cell death. Our results show that EGFR mediates the cytotoxic effects of cyclosporine through an ROS-independent mechanism. Cyclosporine-induced cell death is triggered by a non-classical phosphoinositide 3-kinase and does not require ERK activation.