Background: Lung cancer has the highest mortality rate among cancers worldwide, with non-small cell lung cancer (NSCLC) the most common type. Increasing evidence shows that PHB2 is highly expressed in other cancer types; however, the effects of PHB2 in NSCLC are currently poorly understood. Method: PHB2 expression and its clinical relevance in NSCLC tumor tissues were analyzed using a tissue microarray. The biological role of PHB2 in NSCLC was investigated in vitro and in vivo using immunohistochemistry and immunofluorescence staining, gene expression knockdown and overexpression, cell proliferation assay, flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, wound healing assay, Transwell assay, western blot analysis, qRT-PCR, coimmunoprecipitation, and mass spectrometry analysis. Results: Our major finding is that PHB2 facilitates tumorigenesis in NSCLC by interacting with and stabilizing RACK1, which further induces activation of downstream tumor-promoting effectors. PHB2 was found to be overexpressed in NSCLC tumor tissues, and its expression was correlated with clinicopathological features. Furthermore, PHB2 overexpression promoted proliferation, migration, and invasion, whereas PHB2 knockdown enhanced apoptosis in NSCLC cells. The stimulating effect of PHB2 on tumorigenesis was also verified in vivo. In addition, PHB2 interacted with RACK1 and increased its expression through posttranslational modification, which further induced activation of the Akt and FAK pathways. Conclusions: Our results reveal the effects of PHB2 on tumorigenesis and its regulation of RACK1 and RACK1-associated proteins and downstream signaling in NSCLC. We believe that the crosstalk between PHB2 and RACK1 provides us with a great opportunity to design and develop novel therapeutic strategies for NSCLC.