Background: Currently,EGFR-TKIs are widely accepted as the standard treatment for EGFR-mutant advanced non-small-cell lung cancer (NSCLC); however, acquired resistance is inevitable. Combination therapy is considered as a strategy to overcome the resistance to EGFR-TKIs. Anlotinib, a novel multi-targeting, small-molecule TKI, has shown active to suppress tumor angiogenesis and growth. However, there is still a lack of evidence supporting the use of EGFR-TKIs in combination with anlotinib for the treatment of NSCLC until now. A multi-center, single-arm, phase II clinical trial was therefore designed to examine the efficacy and safety of EGFR-TKIs combined with anlotinib for treatment-naïve, advanced NSCLC patients, and unravel the possible mechanisms. Methods: This study was conducted in 14 research centers in Fujian, China. The main eligibility criteria were stage IV or relapsed nonsquamous NSCLC with EGFR mutations (exon 19 deletion,, and L858R), ECOG score 0-2,and age 20 to 75 years and no previous systemic treatment. Patients with asymptomatic brain metastases were admitted.Eligible patients were given gefitinib (250 mg QD) or icotinib (125 mg TID) in combination with anlotinib (10 mg per day, on days 1‒14; 21 days per cycle) until disease progression. The primary endpoint is progression-free survival (PFS) and safety, and the secondary endpoint is overall survival (OS), objective response rate (ORR) and disease control rate (DCR).Peripheral blood was sampled pre-treatment, once every two months during treatment and after disease progression, andT790M mutation was detected in plasma ctDNA using a droplet digital PCR (ddPCR) assay. Results: Of 60 patients enrolled (August 2, 2018 to May 28, 2020). As of February 1, 2021, 37 patients (61.7%) experienced PFS events and 10 (16.7%) died. The ORR was 78.3%, and the DCR was100%.Median PFS was 13.0 months (95%CI,10.7-15.3).The 5 most common treatment-related adverse events included rash (63.3%), fatigue (55.0%), hypertension (48.3%), diarrhea (33.3%) and hand-foot syndrome (30.0%), and grade 3 adverse events included hypertension (5.0%), rash (1.67%), hypertriglyceridemia (1.67%), vomiting (1.67%) and elevated ALT (1.67%); no grade 4 adverse events or drug-related deaths were observed. Peripheral blood samples were collected from 36 patients pre-treatment, and 30.6% were identified with low-frequency de novo T790M mutations, with the mutation-allele frequency (MAF) ranging from 0.01% to 0.28%. Conclusions: The combination of the first-generation EGFR-TKIs and anlotinib shows impressive ORR and DCR, and acceptable toxicity in treatment-naïve advanced NSCLC patients with activating EGFR mutations, and we observed a high proportion of patients harboring de novo EGFR T790M mutations in this study. Clinical trial information: NCT03720873