Smoking is associated with worse clinical outcomes for lung cancer patients. Cell-based studies suggest that NNK (a tobacco specific carcinogen) promotes lung cancer progression. Given its short half-life, the physiological relevance of these in vitro results remains elusive. NNAL, a major metabolite of NNK with a similar structure, a chiral center, and a longer half-life, has never been evaluated in cancer cells. In this study, we characterized the effect of NNAL and its enantiomers on cancer progression among a panel of NSCLC cell lines and explored the associated mechanisms. We found that (R)-NNAL promotes cell proliferation, enhances migration, and induces drug resistance while (S)-NNAL has much weaker effects. Mechanistically, (R)-NNAL phosphorylates and deactivates LKB1 via the β-AR signaling in the LKB1 wild type NSCLC cell lines, contributing to the enhanced proliferation, migration, and drug resistance. Of note, NNK exposure also increases the phosphorylation of LKB1 in A/J mice. More importantly, human lung cancer tissues appear to have elevated LKB1 phosphorylation. Our results reveal, for the first time, that NNAL may promote lung cancer progression through LKB1 deactivation in an isomer-dependent manner.