Although the role of Yes-associated protein (Yap) has been described in the progression of lung cancer, the downstream effector of the Yap-Hippo pathway has not been identified. Accordingly, the aim of our study is to explore whether Yap modulates the activity of lung cancer by controlling mitochondrial elongation factor 1 (MIEF1)-related mitochondrial stress in a manner dependent on the JNK pathway. Cell viability was determined via MTT, LDH release and immunofluorescence assays. ATP production, the mitochondrial membrane potential, and caspase-9 activity were investigated to assess mitochondrial function. siRNA transfection and pathway blockers were used to observe the roles of MIEF1 and JNK in Yap-modulated cell viability in lung cancer cells in vitro. Yap deletion reduced cell viability in A549 and H358 lung cancer cells. At the molecular level, Yap deletion promoted mitochondrial dysfunction, as evidenced by the decreased mitochondrial potential, increased mitochondrial oxidative stress, augmented mitochondrial pro-apoptotic factor leakage and elevated caspase-9 activity. In addition, we found that Yap modulated mitochondrial stress via MIEF1 and that loss of MIEF1 abolished the regulatory actions of Yap on mitochondrial stress and cell viability. Besides, we provided evidence to support the necessary role of JNK in Yap-mediated MIEF1 upregulation. Inhibition of JNK abolished the promotive effect of Yap deletion on MIEF1 activation. Taken together, our results identified the JNK-MIEF1 pathway and mitochondrial stress as downstream effectors of Yap in lung cancer. This finding suggests a novel approach for the treatment of lung cancer in clinical practice.