Objective Anlotinib, an oral vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor, has confirmed antitumor activity in lung cancer in both in vitro and in vivo assays, and has been recommended as third-line treatment agent in non-oncogene driven non-small cell lung cancer (NSCLC). This prospective study aimed to investigate the efficacy and safety of anlotinib plus S-1 for third- or later-line treatment in patients with advanced NSCLC. Methods Patients with histologically or cytologically confirmed NSCLC, and documented disease progression following second-line chemotherapy, and/or epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) treatment were enrolled in this study. The patients were treated anlotinib (8 mg daily d 1–14) and S-1 (60 mg/m2 d 1–14) and the treatment was repeated every 3 weeks. Treatment was continued until disease progression or unacceptable toxicity occurred. The objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and adverse events (AEs) were reviewed and evaluated. Results Forty-one patients were enrolled in the study between June 2018 and December 2018. The total ORR and DCR were 26.8％ and 80.5％, respectively. The median PFS was 5.2 months [95％ confidence interval (CI), 3.9 to 6.6 months]. In the univariate analysis, there was a significant difference in the median PFS between patients with brain metastases and those without brain metastases (4.8 months vs 5.9 months, respectively; P = 0.039). The East Cooperative Oncology Group (ECOG) performance status (P = 0.002), lines of therapy (P = 0.015), and therapeutic evaluation (P = 0.014) were independent factors that influenced PFS. The most common AEs were hypertension, proteinuria, myelosuppression, gastrointestinal reactions, fatigue, and mucositis.Conclusion Anlotinib plus S-1 is an effective and safe regimen for advanced NSCLC as third- or later-line therapy.