Background: Several studies show that EGFR-mutant NSCLC patients (pts) gained response to EGFR-TKI treatment again after a TKI free interval. To date, no prospective evaluation of the clinical effects of EGFR-TKI re-challenge in EGFR-mutant NSCLC pts has been performed. Methods: This was a multicenter, open-label, single-arm, phase II study (CTONG1304, NCT01933347). Stage IIIB/IV NSCLC pts with EGFR exon 19del/L858R mutation, who previously benefited from first-line gefitinib treatment followed by second-line chemotherapy, took gefitinib 250mg/d until disease progression or death or intolerable toxicity occurred. Blood samples were dynamically collected for EGFRmutation testing using droplet digital PCR at every visit (from baseline to the end of gefitinib treatment). The primary objective was disease control rate (DCR) at week 8. Secondary objectives were objective response rate (ORR), progression free survival (PFS), and overall survival (OS). Results: From March 2014 to May 2016, 45 eligible pts were enrolled and 43 pts were included in the full analysis set (FAS) for efficacy analysis. Gefitinib re-challenge achieved DCR of 69.8%. ORR was 4.7%. Median PFS and OS were 4.4 and 8.0 months (m) respectively. T790M- subgroup at baseline had higher DCR, longer mPFS and mOS, compared with T790M+ subgroup. EGFRstatus changed significantly after gefitinib re-challenge. Conclusions: Gefitinib re-challenge was an effective option in EGFR-mutant NSCLC pts. T790M negativity is a potentially predictive efficacy biomarker for gefitinib re-challenge. Clinical trial information: NCT01933347