Mammalian STE20-like kinase 1 (Mst1) is well recognized as a major tumor suppressor in cancer development, growth, metabolic reprogramming, metastasis, cell death and recurrence. However, the roles of Mst1 in non-small cell lung cancer (NSCLC) A549 cell phenotypic alterations remain to be elucidated. The present study aimed to explore the functional role and underlying mechanisms of Mst1 with regards to A549 cell proliferation, migration and apoptosis; this study focused on mitochondrial homeostasis and Rho-associated coiled-coil containing protein kinase 1 (ROCK1)/F-actin pathways. The results demonstrated that Mst1 was downregulated in A549 cells compared with in a normal pulmonary epithelial cell line. Subsequently, overexpression of Mst1 in A549 cells reduced cell viability and promoted cell apoptosis. Furthermore, overexpression of Mst1 suppressed A549 cell proliferation and migration. At the molecular level, the reintroduction of Mst1 in A549 cells led to activation of mitochondrial apoptosis, as evidenced by a reduction in mitochondrial potential, overproduction of ROS, cytochrome c release from the mitochondria into the nucleus, and upregulation of pro-apoptotic protein expression. In addition, Mst1 overexpression was closely associated with impaired mitochondrial respiratory function and suppressed cellular energy metabolism. Functional studies illustrated that Mst1 overexpression activated ROCK1/F-actin pathways, which highly regulate mitochondrial function. Inhibition of ROCK1/F-actin pathways in A549 cells sustained mitochondrial homeostasis, alleviated caspase-9-dependent mitochondrial apoptosis, enhanced cancer cell migration and increased cell proliferation. In conclusion, these data firmly established the regulatory role of Mst1 in NSCLC A549 cell survival via the modulation of ROCK1/F-actin pathways, which may provide opportunities for novel treatment modalities in clinical practice.