NSCLC is a heterogeneous disease with unique combinations of somatic molecular alterations in individual patients. The different mutations in tumor oncogene and suppressors might be associated with the response to therapy. However, little is known about how Smad4 genomic alterations cause the therapeutic effect of docetaxel. The retrospective analysis was conducted on 49 patients with stage IIB or IIIA non-small cell lung cancer receiving docetaxel chemotherapy. One novel missense variant, c.1060 G > C in Smad4 was identified by next-generation sequencing. The Smad4c.1060 G > C variant results in the substitution of valine with leucine at amino acid 354 (p.Val354Leu, V354L). The clinical analysis showed that the patients with Smad4 V354L mutation receiving docetaxel treatment manifested better prognosis with prolonged disease-free survival and overall survival compared with patients with the wild-type. Smad4 V354L cells demonstrated increased sensitivity to docetaxel with apoptosis and G2/M cell cycle arrest. Furthermore, the cell-cycle related protein expression of CDK2 was remarkably decreased, while the expression of cyclin-dependent kinase inhibitor p21 Waf1 and p27 Kip1 was significantly increased. In vivo experiments further demonstrated the increased inhibitory effects of docetaxel in the nude mice inoculated with Smad4 V354L cells compared to the mice inoculated with wild-type cells group. The novel V354L missense mutation of Smad4 gene enhances the efficacy of docetaxel in non-small cell lung cancer, which would provide new opportunities for precise clinical therapy of NSCLC.