Chemoresistance is an obstacle to the successful treatment of nasopharyngeal carcinoma (NPC). To explore novel treatment strategies, we first characterized the lapatinib-sensitivity of a panel of NPC cell lines by SRB and clonogenic cytotoxic assays and found that the highly metastatic NPC (C666-1 and 5-8F) cells are significantly more resistant and the poorly metastatic lines (6-10B, TW01 and HK-1) more sensitive to lapatinib. Western blot analysis of the lapatinib-sensitive 6-10B and resistant 5-8F NPC cells showed that the expression of phosphorylated/inactive FOXO3(P-FOXO3;T32), its target FOXM1 and its regulator SIRT2 correlates negatively with lapatinib response and sensitivity, suggesting that SIRT2 mediates FOXO3 deacetylation to promote lapatinib resistance. In agreement, clonogenic cytotoxic assays using wild-type and foxo1/3/4−/− MEFs showed that FOXO1/3/4-deletion significantly attenuates lapatinib-induced cytotoxicity, confirming that FOXO proteins are essential for mediating lapatinib response. SRB cell viability assays using chemical SIRT inhibitors (i.e. sirtinol, Ex527, AGK2 and AK1) revealed that all SIRT inhibitors can reduce NPC cell viability, but only the SIRT2-specific inhibitors AK1 and AGK2 further enhance the lapatinib cytotoxicity. Consistently, clonogenic assays demonstrated that the SIRT2 inhibitors AK1 and AGK2 as well as SIRT2-knockdown increase lapatinib cytotoxicity further in both the sensitive and resistant NPC cells. Co-immunoprecipitation studies showed that besides lapatinib treatment, SIRT2-pharmaceutical inhibition and silencing also led to an increase in FOXO3 acetylation. Importantly, SIRT2 inhibition and depletion further enhanced lapatinib-mediated FOXO3-acetylation in NPC cells. Collectively, our results suggest the involvement of SIRT2-mediated FOXO3 deacetylation in lapatinib response and sensitivity, and that SIRT2 can specifically antagonise the cytotoxicity of lapatinib through mediating FOXO3 deacetylation in both sensitive and resistant NPC cells. The present findings also propose that SIRT2 can be an important biomarker for metastatic and lapatinib resistant NPC and that targeting the SIRT2-FOXO3 axis may provide novel strategies for treating NPC and for overcoming chemoresistance.