Gentamicin is a nephrotoxic antibiotic that causes acute kidney injury (AKI) primarily by targeting the proximal tubule epithelial cell. The development of an effective therapy for gentamicin-induced renal cell injury is limited by incomplete mechanistic insight. To address this challenge, we propose that RNAi signal pathway screening could identify a unifying mechanism of gentamicin-induced cell injury and suggest a therapeutic strategy to ameliorate it. Computational analysis of RNAi signal screens in gentamicin-exposed human proximal tubule cells suggested the cross-organelle stress response (CORE), the unfolded protein response (UPR), and cell chaperones as key targets of gentamicin-induced injury. To test this hypothesis, we assessed the effect of gentamicin on the CORE, UPR, and cell chaperone function, and tested the therapeutic efficacy of enhancing cell chaperone content. Early gentamicin exposure disrupted the CORE, evidenced by a rise in the ATP:ADP ratio, mitochondrial-specific H2O2 accumulation, Drp-1-mediated mitochondrial fragmentation, and endoplasmic reticulum-mitochondrial dissociation. CORE disruption preceded measurable increases in whole-cell oxidative stress, misfolded protein content, transcriptional UPR activation, and its untoward downstream effects: CHOP expression, PARP cleavage, and cell death. Geranylgeranylacetone, a therapeutic that increases cell chaperone content, prevented mitochondrial H2O2 accumulation, preserved the CORE, reduced the burden of misfolded proteins and CHOP expression, and significantly improved survival in gentamicin-exposed cells. We identify CORE disruption as an early and remediable cause of gentamicin proteotoxicity that precedes downstream UPR activation and cell death. Preserving the CORE significantly improves renal cell survival likely by reducing organelle-specific proteotoxicity during gentamicin exposure.