Rho-associated kinase 1 (ROCK1) regulates tumor metastasis by maintaining cellular cytoskeleton homeostasis. However, the precise role of ROCK1 in non-small-cell lung cancer (NSCLC) apoptosis remains largely unknown. In this study, we examined the function of ROCK1 in NSCLS survival using RNA interference-mediated knockdown. Our results showed that ROCK1 knockdown reduced A549 lung cancer cell viability in vitro. It also inhibited A549 cell migration and proliferation. Transfection of ROCK1 siRNA was associated with increased expression of large tumor suppressor kinase 2 (LATS2) and c-Jun N-terminal kinase (JNK). Moreover, ROCK1 knockdown-induced A549 cell apoptosis and inhibition of proliferation were suppressed by LATS2 knockdown or JNK inactivation, suggesting that ROCK1 deficiency triggers NSCLC apoptosis in a LATS2-JNK pathway-dependent manner. Functional analysis further demonstrated that ROCK1 knockdown dysregulated mitochondrial dynamics and inhibited mitochondrial biogenesis. This effect too was reversed by LATS2 knockdown or JNK inactivation. We have thus identified a potential pathway by which ROCK1 downregulation triggers apoptosis in NSCLC by inducing LATS2-JNK-dependent mitochondrial damage.