Melamine causes renal tubular cell injury through inflammation, fibrosis and apoptosis. While melamine effects the rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i), reactive oxygen species (ROS) production, and pro-apoptotic pathway activation, the mechanism of upstream Ca²⁺ signaling is unknown. Since melamine has some structural similarities with L-amino acids, which endogenously activates Ca²⁺-sensing receptors (CSR), we examined the effect of melamine on CSR-induced Ca²⁺ signaling and apoptotic cell death. We show here that melamine activates CSR, causing a sustained Ca²⁺ entry in renal epithelial cell line, LLC-PK1. Moreover, such CSR stimulation resulted a rise in [Ca²⁺]_i, leading to enhanced ROS production. Furthermore, melamine-induced elevated [Ca²⁺]_i and ROS production caused a dose dependent increase in apoptotic (by DAPI staining, DNA laddering, Annexin V assay) and necrotic (propidium iodide staining) cell death. Upon examining the downstream mechanism, we found that transforming growth factor β1 (TGFβ1), which increases extracellular matrix genes and pro-apoptotic signaling were also up-regulated at lower doses of melamine, which could be due to an early event inducing apoptosis. Additionally, cells exposed to melamine displayed a rise in phospho-extracellular signal-regulated kinase (pERK) activation and lactate dehydrogenase (LDH) release resulting in cytotoxicity. These results offer a novel insight into the molecular mechanisms by which melamine exerts its effect on CSR, causing a sustained elevation of [Ca²⁺]_i, leading to ROS generation, fibronectin production, pro-apoptotic pathway activation and renal cell damage. Together, these results thus suggest that melamine-induced apoptosis and/or necrosis may subsequently results in acute kidney injury (AKI) and promote kidney stone formation.