The environmental pollutant cadmium (Cd) poses a threat to human health through the consumption of contaminated foodstuffs culminating in chronic nephrotoxicity. Mitochondrial dysfunction and excessive reactive oxygen species (ROS) are key to Cd cellular toxicity. Cd-lipid interactions have been less considered. We hypothesized Cd binding to the inner mitochondrial membrane (IMM) phospholipid cardiolipin (CL) and membrane rigidification underlies defective electron transfer by disrupted respiratory supercomplexes (SCs). In Cd-treated rat kidney cortex (rKC) mitoplasts, laurdan (lipid-water interface), and diphenylhexatriene (hydrophobic core) revealed increased and decreased membrane fluidity, respectively. Laurdan-loaded pure CL or IMM biomimetic (40 mol % POPC, 35 mol % DOPE, 20 mol % TOCL, 5 mol % SAPI) nanoliposomes were rigidified by 25 μM Cd, which was confirmed in live-cell imaging of laurdan or di-4-ANEPPDHQ loaded human proximal convoluted tubule (HPCT) cells. Blue native gel electrophoresis evidenced ∼30% loss of I+III