Purpose: Anlotinib is an anti-angiogenetic multi-targeted tyrosine kinase inhibitor. This study aimed to evaluate the efficacy and safety of anlotinib in advanced non-small cell lung cancer (aNSCLC) in the real world.Methods: Patients with aNSCLC receiving anlotinib were enrolled in two cohorts (treatment-naive and previously treated). The endpoints included progression-free survival (PFS), overall survival (OS) and anlotinib-related adverse events (ar-AEs).Results: 203 patients accrued in the study. In the treatment-naïve cohort (n=80), the PFS was 7.4 (95% confidence interval [CI], 4.1-10.7) and OS was 10.8 (95% CI, 5.8-15.8) months of monotherapy group (immature survival for combination group). In previously treated cohort (n=123), The PFS was 8.0 months (95% CI, 6.1-9.9) in the combination group and 4.3 months (95% CI, 2.1-6.6) in the monotherapy group (hazard ratio [HR], 0.49; 95% CI, 0.29-0.83; p=0.007), respectively. The OS was 18.5 months (95% CI, 10.5- to 26.6) in the combination group and 7.8 months (95% CI, 7.1-8.4) in the monotherapy group (HR, 0.38; 95% CI, 0.22-0.66; p=0.001), respectively. The ar-AEs of grade ≥3 in the monotherapy and the combination groups were hypertension (9.0% and 8.7%), fatigue (8.1% and 7.6%), hand-foot syndrome (8.1% and 6.5%), diarrhea (5.4% and 8.7%), proteinuria (5.4% and 5.4%), and mucositis oral (6.3% and 8.7%).Conclusion: In aNSCLC, anlotinib monotherapy has a promising efficacy in the first-line setting. It may be an option for those who are ineligible for chemotherapy; anlotinib combination therapy in a ≥ second-line setting showed manageable toxicities and encouraging efficacy, indicating a good application prospect.Trial registration: This study was retrospectively registered with ISRCTN Registry (ID ISRCTN35543977) on January 26th, 2021 and Chinese Clinical Trial Register (ChiCTR2000032265) on April 4th, 2020.