<p>Patients with lung cancer harboring a KRAS oncogenic driver mutation have a very poor prognosis. Recently, we reported that SIRT1 is upregulated by the KRAS^Mut–c-Myc axis, and that KRAS^Mut-induced SIRT1 is stably deacetylated at lysine 104, which in turn increases KRAS^Mut activity and enhances chemoresistance. Notably, SIRT1 activity as well as SIRT1 levels are more elevated in KRAS^Mut cells compared with EGFR^Mut, KRAS^Mut- and EGFR^Mut-negative cells, and nontumorigenic cells. This prompted us to investigate the mechanism by which SIRT1 activity was increased and the role of pSIRT1 in the chemoresistance of KRAS^Mut lung cancer cells. The activated MEK–ERK pathway under KRAS^Mut increased AP-1 transcription activity, which in turn enhanced TGF-β1 secretion. The secreted TGF-β1 activated the Smad2/3–JNK1 signaling pathway in an autocrine manner, increasing pSIRT1^S27 and pSIRT1^S47, ultimately enhancing KRAS^Mut activity through KRAS deacetylation and affecting chemoresistance. We identified a small molecule from the natural compound library—Kuwanon C (KWN-C), a SIRT1 activity inhibitor—which reduced pSIRT1^S27 and pSIRT1^S47 levels via a decrease in the activity of the TGF-β1–-Smad2/3–JNK1 signaling pathway. Treatment with the SIRT1 activity inhibitor triggered the anticancer effects of cisplatin and pemetrexed in human lung cancer cells, lung orthotopic tumors and a spontaneous in vivo model of KRAS^Mut lung cancer. Our findings reveal a novel pathway critical for the regulation of SIRT1 activity in KRAS^Mut lung cancer and provide important evidence for the potential application of SIRT1 activity inhibitors as an adjuvant chemotherapy, overcoming chemoresistance in patients with KRAS^Mut lung cancer.</p>