SMAD4 is an intracellular signaling mediator of the TGF-β pathway. Its mutation was commonly observed in gastrointestinal cancers, such as pancreatic cancer. The loss of SMAD4 on immunohistochemical staining is often used to suggest a pancreaticobiliary differentiation in evaluating a metastatic adenocarcinoma with unknown origin. However, the function and molecular mechanism of SMAD4 in non-small cell lung cancer (NSCLC) development are largely unknown. Thus, we studied the correlation between SMAD4 mutations and clinico-molecular features in the patients with NSCLC. We reported the frequencies and prognostic values of SMAD4 mutations in a Chinese NSCLC cohort using next-generation sequencing. The NSCLC cases from several public databases, including The Cancer Genome Atlas and others, were also used in this study to elucidate SMAD4-related molecular partners and mechanisms. Integrated bioinformatics analyses were conducted, such as analysis of Gene Ontology enrichment analysis, gene set enrichment analysis (GSEA), and survival analysis. Immunohistochemistry showed that the tissues harboring SMAD4 mutations tended to show SMAD4 deficiency or loss, while SMAD4 expression was significantly reduced at all stages of NSCLC cases. We found that reduced SMAD4 expression was more frequent in the patients with poor disease-free survival and resistance to platinum-based chemotherapy. SMAD4 mutation was an independent risk factor for the survival of NSCLC patients. The expression of SMAD4 was associated with that of SMAD2. The GSEA showed that SMAD4 might promote NSCLC progression by regulating proliferation, adhesion, and immune response. In conclusion, these data suggest that SMAD4 mutation or loss as well as reduced expression can be used to identify the NSCLC patients with poor survival and resistance to platinum-based chemotherapy. SMAD4 may be a predictive marker or therapeutic target in NSCLC. The source code and user's guide are freely available at Github: https://github.com/wangyue77-ab/smad4 .