EGFR kinase inhibitors including gefitinib and erlotinib exert potent therapeutic efficacy in non-small cell lung cancers harboring EGFR activating mutations. However, most patients ultimately develop resistance to these drugs. Here we report a novel mechanism of acquired resistance the reversal of which could clinical outcomes. In erlotinib-resistant lung cancer cells harboring activating EGFR mutations that we established, there was increased expression of Src, integrinβ1, α2, and α5 along with enhanced cell adhesion activity. Interestingly, RNAi-mediated silencing of integrinβ1 restored erlotinib sensitivity and reduced activation of Src and Akt after erlotinib treatment. Further, Src silencing inhibited Akt phosphorylation and cell growth, with this inhibitory effect further augmented by erlotinib treatment. Increased expression of integrinβ1, α5, and/or α2 was also observed in refractory tumor samples from lung cancer patients treated with erlotinib and/or gefitinib. Together, our findings identify the integrinβ1/Src/Akt signaling pathway as a key mediator of acquired resistance to EGFR-targeted anticancer drugs.