Lung cancer is the leading cause of cancer-related mortality worldwide. Non-small cell lung cancer (NSCLC) is the most common pathological subtype of lung cancer and is associated with low 5-year overall survival rates. Therefore, novel and effective chemotherapeutic drugs are urgently required for improving the survival outcomes of patients with lung cancer. Cyclovirobuxine D (CVB-D) is a natural steroidal alkaloid, used for the treatment of cardiovascular diseases in Traditional Chinese Medicine. Several studies have also demonstrated the antitumor effects of CVB-D. Therefore, in the present study, the therapeutic effects of CVB-D in lung cancer and the underlying mechanisms were investigated using the in vivo xenograft model of NSCLC in nude mice and in vitro experiments with the NSCLC cell lines. Bioinformatics analyses of RNA-sequencing data, and cell-based functional assays demonstrated that CVB-D treatment significantly inhibited in vitro and in vivo NSCLC cell proliferation, survival, invasion, migration, angiogenesis, epithelial-to-mesenchymal transition and G(2)/M phase cell cycle. CVB-D exerted its antitumor effects by inhibiting the KIF11-CDK1-CDC25C-cyclinB1 G(2)/M phase transition regulatory oncogenic network and the NF-kappa B/JNK signaling pathway. CVB-D treatment significantly reduced the sizes and weights and malignancy of xenograft NSCLC tumors in the nude mice. In conclusion, the present study demonstrated that CVB-D inhibited the growth and progression of NSCLC cells by inhibiting the KIF11-CDK1-CDC25C-CyclinB1 G(2)/M phase transition regulatory network and the NF-kappa B/JNK signaling pathway. Therefore, CVB-D is a promising drug for the treatment of NSCLC patients.