Smoking has been identified as a major risk factor for the development and progression of non-small cell lung cancer (NSCLC). As a key component of tobacco smoke, nicotine is believed to play a significant role in promoting NSCLC growth and progression. EZH2 is an epigenetic regulator highly expressed in the tumor tissues of smokers. However, whether and how nicotine regulates the expression of EZH2 and the underlying mechanisms remain unclear. Bioinformatics analysis and immunohistochemistry were used to compare the expression of EZH2 in NSCLC samples between smokers and nonsmokers. Western blotting, real-time quantitative PCR, and immunofluorescence were employed to confirm the effects of nicotine on EZH2 expression. Cell Counting Kit-8 assays, colony formation assays, 5-ethynyl-2-deoxyuridine staining, and Transwell assays were conducted to analyze the proliferation and metastasis of A549 and H1650 cells treated with siRNA or EZH2 inhibitors. Real-time quantitative PCR and chromatin immunoprecipitation assays were performed to assess the regulatory effect of nicotine on EZH2 transcript levels via c-Myc. Coimmunoprecipitation and ubiquitination assays were used to assess the deubiquitination of c-Myc by OTUB1. Finally, a nude mouse model was used to evaluate the impact of combined c-Myc and EZH2 inhibitors on tumor proliferation and metastasis in vivo. EZH2 is expressed at relatively high levels in NSCLC patients, as determined by both bioinformatic and IHC analyses. Nicotine upregulates EZH2 expression and promotes the proliferation and metastatic ability of lung cancer cells. Inhibition of EZH2 with either DZNep or EPZ6438, EZH2 inhibitors, or siRNA significantly decreased the proliferative and metastatic capacity of NSCLC cells induced by nicotine treatment. Moreover, the study revealed that nicotine induces OTUB1 expression, stabilizes the c-Myc protein via deubiquitination, and enables c-Myc-mediated transcriptional activation of EZH2. Furthermore, the c-Myc inhibitor 10058-F4 exhibited synergistic effects with the EZH2 inhibitor DZNep in suppressing NSCLC cell proliferation and metastasis both in vitro and in vivo.Nicotine regulates the c-Myc/EZH2 signaling pathway via OTUB1-mediated deubiquitination, thereby promoting the proliferation and metastasis of NSCLC cells. This research reveals novel molecular mechanisms of nicotine in the development of NSCLC, providing a theoretical foundation for future therapeutic strategies.