The promising therapeutic efficacy of the third generation EGFR inhibitor, osimertinib (AZD9291), for the treatment of patients with EGFR-mutant non-small cell lung cancer (NSCLC) has been demonstrated in the clinic both as first-line and second line therapy. However, inevitable acquired resistance limits its long-term benefit to patients and is thus a significant clinical challenge. The current study focuses on studying the potential role of targeting MEK5-ERK5 signaling in overcoming acquired resistance to osimertinib. Osimertinib and other third generation EGFR inhibitors exerted a rapid and sustained suppressive effect on ERK5 phosphorylation primarily in EGFR-mutant NSCLC cell lines and lost this activity in some osimertinib-resistant cell lines. Osimertinib combined with either ERK5 or MEK5 inhibitors synergistically decreased the survival of osimertinib-resistant cell lines with enhanced induction of apoptosis primarily via augmenting Bim expression. Moreover, the combination effectively inhibited the growth of osimertinib-resistant xenografts in vivo. Together, these findings suggest the potential role of MEK5-ERK5 signaling in modulating development of acquired resistance to osimertinib and value of targeting this signaling as a potential strategy in overcoming acquired resistance to osimertinib and possibly other third generation EGFR inhibitors.