Afatinib, an irreversible ErbB-family blocker, could improve the survival of advanced epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer patients (NSCLCm+). This phase II trial (NCT04201756) aimed to assess the feasibility of neoadjuvant Afatinib treatment for stage III NSCLCm+. Forty-seven patients received neoadjuvant Afatinib treatment (40 mg daily). The primary endpoint was objective response rate (ORR). Secondary endpoints included pathological complete response (pCR) rate, pathological downstaging rate, margin-free resection (R0) rate, event-free survival, disease-free survival, progression-free survival, overall survival, treatment-related adverse events (TRAEs). The ORR was 70.2% (95% CI: 56.5% to 84.0%), meeting the pre-specified endpoint. The major pathological response (MPR), pCR, pathological downstaging, and R0 rates were 9.1%, 3.0%, 57.6%, and 87.9%, respectively. The median survivals were not reached. The most common TRAEs were diarrhea (78.7%) and rash (78.7%). Only three patients experienced grade 3/4 TRAEs. Biomarker analysis and tumor microenvironment dynamics by bulk RNA sequencing were included as predefined exploratory endpoints. CISH expression was a promising marker for Afatinib response (AUC = 0.918). In responders, compared to baseline samples, increasing T-cell- and B-cell-related features were observed in post-treatment tumor and lymph-node samples, respectively. Neoadjuvant Afatinib is feasible for stage III NSCLC+ patients and leads to dynamic changes in the tumor microenvironment.