Background: BRAF variants were reported resistant mechanisms to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC). However, concomitant somatic variations other than BRAF variants and efficacy of subsequent treatment remained unclear. Methods: From October 2016 to May 2020, advanced NSCLC patients who underwent next-generation sequencing and were detected with co-mutation of BRAF and EGFR activating mutations are retrospectively included. Since June 2020, EGFR-mutant patients with acquired BRAF V600E after progression from previous Osimertinib are prospectively arranged to explore efficacy of the EGFR-BRAF co-inhibition. Results: Fifty-eight patients were retrospectively identified and five patients were prospectively included. BRAF variants was acquired after a median time of 22.7 months from initial diagnosis. Variations of TP53, PIK3CA, RB1, MET, LRP1B, APC, CDKN2A, MYC, ERBB2 and SMAD4 were over 10%, which were enriched in cell cycle/p53 pathway, EGFR downstream and bypass pathway. Subsequently, median progression-free survival was 5.0 months for chemotherapy and 2.1 months for TKI treatment without targeting both EGFR and BRAF respectively (p = 0.019). Osimertinib plus vemurafenib (n = 4) or dabrafenib + trametinib (n = 1) was prospectively administrated in five patients. Median PFS was 7.8 months. Grade 3 rash was observed in one patient. Upon disease progression, activation of RAS signaling was observed. Conclusions: Variations of EGFR downstream or bypass pathway were also frequent in patient with co-mutation of EGFR and BRAF. Efficacy of subsequent TKI without targeting both EGFR and BRAF was inferior to chemotherapy. EGFR-BRAF co-inhibition showed improved efficacy. More treatment strategy should be explored in the future.