Despite notable advances in chemotherapy protocols and targeted therapies, ensuing drug resistance limits the efficacy of cancer treatments, calling for the identifying druggable targets that can overcome chemo-resistance. Here we show that the mitochondria-shaping protein OPA1 takes part in the resistance against the tyrosine kinase inhibitor gefitinib in lung adenocarcinoma cells. In gefitinib-resistant lung cancer cells, OPA1 levels were increased, mitochondrial cristae structures were narrower and mitochondrial respiration increased. Genetic and pharmacological OPA1 inhibition in the resistant lung cancer cells sensitized them to gefitinib-induced cytochrome c release and apoptosis. In vivo, orthotopic tumors formed by the injection of gefitinib-resistant lung cancer cells were insensitive to gefitinib treatment, but a combination of gefitinib and OPA1 inhibitor reduced tumor size and increased apoptosis. Our data identify the mitochondrial protein OPA1 as a downstream factor that sustains gefitinib resistance and can be targeted to overcome chemo-resistance.