Intracellular Ca²⁺ is a key regulator of life or death decisions in cultured neurons and sensory cells. The role of Ca²⁺ in these processes is less clear in vivo, as the location of these cells often impedes visualization of intracellular Ca²⁺ dynamics. We generated transgenic zebrafish lines that express the genetically encoded Ca²⁺ indicator GCaMP in mechanosensory hair cells of the lateral line. These lines allow us to monitor intracellular Ca²⁺ dynamics in real time during aminoglycoside-induced hair cell death. After exposure of live larvae to aminoglycosides, dying hair cells undergo a transient increase in intracellular Ca²⁺ that occurs shortly after mitochondrial membrane potential collapse. Inhibition of intracellular Ca²⁺ elevation through either caged chelators or pharmacological inhibitors of Ca²⁺ effectors mitigates toxic effects of aminoglycoside exposure. Conversely, artificial elevation of intracellular Ca²⁺ by caged Ca²⁺ release agents sensitizes hair cells to the toxic effects of aminoglycosides. These data suggest that alterations in intracellular Ca²⁺ homeostasis play an essential role in aminoglycoside-induced hair cell death, and indicate several potential therapeutic targets to stem ototoxicity.