Cd²⁺ induces apoptosis of kidney proximal tubule (PT) cells. Mitochondria play a pivotal role in toxic compound-induced apoptosis by releasing cytochrome c. Our objective was to investigate the mechanisms underlying Cd²⁺-induced cytochrome c release from mitochondria in rat PT cells. Using Hoechst 33342 or MTT assay, 10 μM Cd²⁺ induced ∼5–10% apoptosis in PT cells at 6 and 24 h, which was associated with cytochrome c and apoptosis-inducing factor release at 24 h only. This correlated with previously described maximal intracellular Cd²⁺ concentrations at 24 h, suggesting that elevated Cd²⁺ may directly induce mitochondrial liberation of proapoptotic factors. Indeed, Cd²⁺ caused swelling of energized isolated kidney cortex mitochondria (EC₅₀ ∼9 μM) and cytochrome c release, which were independent of permeability transition pore (PTP) opening since PTP inhibitors cyclosporin A or bongkrekic acid had no effect. On the contrary, Cd²⁺ inhibited swelling and cytochrome c release induced by PTP openers (PO₄³⁻ or H₂O₂+Ca²⁺). The mitochondrial Ca²⁺ uniporter (MCU) played a key role in mitochondrial damage: 1) MCU inhibitors (La³⁺, ruthenium red, Ru360) prevented swelling and cytochrome c release; and 2) ruthenium red attenuated Cd²⁺ inhibition of PO₄³⁻-induced swelling. Using the Cd²⁺-sensitive fluorescent indicator FluoZin-1, Cd²⁺ was also taken up by mitoplasts. The aquaporin inhibitor AgNO₃ abolished Cd²⁺-induced swelling of mitoplasts. This could be partially mediated by activation of the mitoplast-enriched water channel aquaporin-8. Thus cytosolic Cd²⁺ concentrations exceeding a certain threshold may directly cause mitochondrial damage and apoptotic development by interacting with MCU and water channels in the inner mitochondrial membrane.