Crizotinib, a multi-targeted MET/ALK/ROS1 tyrosine kinase inhibitor, has been approved for the treatment of ROS1 fusion-positive NSCLCs. However, "on-target" or "off-target" resistance alterations often emerge which confer the drug resistance. Patients with ROS1-rearranged NSCLC who develop crizotinib resistance, especially those acquiring "off-target" resistance mutations, still lack effective therapeutic options for post-crizotinib treatment. Herein, we reported a stage IVb lung adenocarcinoma patient harboring ROS1 fusion, who acquired a BRAF V600E and lost the ROS1 fusion after progression on crizotinib. It was deduced that the V600E may originate from a sub-clone with an extremely low fraction that was independent of ROS1 fusion-positive cells. The patient was subsequently treated with dabrafenib and trametinib combination and achieved a partial response lasting for more than 6 months. Our study revealed that BRAF V600E can confer the crizotinib resistance in ROS1 fusion-positive NSCLC and presented the first case showing that the treatment with dabrafenib and trametinib can serve as an effective option for later-line treatment for this molecular-defined subgroup. KEY POINTS: Patients with ROS1-rearranged NSCLC who acquire "off-target" resistance mutations to crizotinib, still lack effective therapeutic options for post-crizotinib treatment. This report describes a case of ROS1-rearranged NSCLC who acquired a BRAF V600E and lost the ROS1 fusion after crizotinib failure. The case was subsequently treated with dabrafenib and trametinib combination and achieved a partial response lasting for more than 6 months. This is the first case reporting the treatment with dabrafenib and trametinib may serve as an effective option for later-line treatment for patients harboring resistant BRAF V600E.© AlphaMed Press 2021.