Objective: To investigate the possibility of the Protein Phosphatase 2A (PP2A) inhibitor, LB100, in reversing acquired resistance to gefitinib in lung adenocarcinoma with epidermal growth factor receptor (EGFR) gene mutation. Methods: Cell line NCI-H1975 and established primary culture cell line 44-1 with gefitinib resistance were sequenced to determine the mutation type of EGFR gene. Cells were treated with gefitinib alone or combined with LB100 to determine the half maximal inhibitory concentration (IC50), and sensitivity of 44-1 and NCI-1975 to gefitinib alone or combined with LB100 was compared. The volume of NCI-H1975 xenografts with different drug treatments was observed to determine the efficiency of gefitinib with or without LB100 in tumor growth inhibition. Results: Both 44-1 and NCI-1975 cells had double EGFR mutation (sensitive L858R mutation and resistant T790M mutation). Both cells showed significant gefitinib resistance (IC50: 23.0 μmol/L in 44-1, 16.7 μmol/L in NCI-1975). When combined with LB100, IC50 of gefitinib decreased to 6.9 μmol/L in 44-1 cell and decreased to 3.4 μmol/L in NCI-H1975 cells. In NCI-1975 xenografts experiments, LB100 enhanced the ability of gefitinib in tumor growth inhibition (P<0.05). Conclusion: LB100 reverses acquired resistance to gefitinib in lung adenocarcinoma cell lines.