<p>Several clinical studies have suggested the benefits of Traditional Chinese Medicine in non-small-cell lung cancer. Ruscogenin is an anti-inflammatory steroid isolated from the root of <i>Ophiopogon japonicus</i> (Thunb.) Ker Gawl., Asparagaceae, has cytotoxic and antitumor potential. A xenograft mouse model was established by subcutaneously injecting A549 cells into mouse’s right axilla. Representative images of tumors and tumor volume were obtained. The levels of proliferating cell nuclear antigen and p38 mitogen-activated protein kinase in tumors were measured by immunohistochemistry staining and immunofluorescence staining, respectively. <i>In vitro</i>, the role of ruscogenin in non-small-cell lung cancer was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, colony formation assays, flow cytometry, wound healing assays, transwell assays, and western blotting. The modulatory function of ruscogenin in p38 mitogen-activated protein kinase signaling was detected by western blotting. Ruscogenin inhibited cell proliferative and metastasis. Additionally, non-small-cell lung cancer cell apoptosis was facilitated by ruscogenin. For <i>in vivo</i> analysis, ruscogenin treatment blocked tumor growth and proliferation. Mechanistically, ruscogenin activated p38 signaling in non-small-cell lung cancer. In conclusion, ruscogenin prevents cell growth and metastasis and promotes cell apoptosis, and inhibits tumor growth in non-small-cell lung cancer by activating p38 mitogen-activated protein kinase signaling.</p> Graphical Abstract <p></p>