This study aimed to investigate the effects and mechanisms of Bozhou Platycodonis Radix extract (PRE) on non-small cell lung cancer (NSCLC) cells. In the experiments, human lung cancer cell line A549 and human lung fibroblast cell line HLF were treated with different concentrations of PRE.. The impacts on cell proliferation, migration, and invasion abilities were evaluated through MTT assay, colony formation assay, cell wound-healing assay, and Transwell assay. Western blot was used to detect the expression of epithelial-mesenchymal transition (EMT) and Ras homolog gene family, member A (RhoA)/Rho-associated coiled-coil forming kinase (ROCK) pathway-related proteins. Additionally, a nude mouse xenograft tumor experiment was conducted to observe the effects of different doses of PRE and lysophosphatidic acid (LPA) injection on tumor volume and weight. The results showed that PRE could significantly inhibit the viability, colony number, migration rate, and the number of cells passing through the membrane of NSCLC cells, and down-regulate the expressions of related proteins in a dose-dependent manner. In contrast, LPA intervention produced the opposite effects. The nude mouse experiment further confirmed the inhibitory effect of PRE. Therefore, PRE may exert anti-NSCLC effects by inhibiting the RhoA/ROCK signaling pathway and the EMT process.