Mitochondria are the organelle responsible for energy production even in cancer cells; however, their roles in chemoresistance remain unclear. Here we show that mitochondrial fusion factor OPA1 is required to maintain the resistance against gefitinib, an EGFR tyrosine kinase inhibitor, in lung adenocarcinoma cells (LUADs). The gefitinib-resistant LUADs highly expressed OPA1, resulting in the elongation of mitochondria with abnormal cristae structure. The overexpressed OPA1 conferred enhanced mitochondrial respiration and anti-apoptotic activity to gefitinib-resistant LUADs. Genetic knockdown or pharmacological inhibition of OPA1 restored the gefitinib sensitivity and induced apoptosis in the resistant LUADs. Furthermore, inhibition of mitochondrial respiration efficiently restored their gefitinib-sensitivity. Thus, inner mitochondrial protein OPA1 and mitochondrial respiration are the downstream factors to sustain the gefitinib resistance. Our results suggest that OPA1 may serve as a novel therapeutic target to overcome chemoresistance in LUADs.