Nicotine is a component of cigarette smoke and mounting evidence suggests toxicity and carcinogenicity of tobacco smoke in kidney. Carcinogenicity of nicotine itself in kidney and the underlying molecular mechanisms are not well-understood. Hence, the objective of this study was to determine the carcinogenic effects of chronic nicotine exposure in Hk-2 human kidney epithelial cells. The effects of nicotine exposure on the expression of genes for cellular reprogramming, redox status, and growth signaling pathways were also evaluated to understand the molecular mechanisms. Results revealed that chronic exposure to nicotine induced growth and neoplastic transformation in HK-2 cells. Increased levels of intracellular reactive oxygen species (ROS), acquired stem cell-like sphere formation, and epithelial-mesenchymal-transition (EMT) changes were observed in nicotine exposed cells. Treatment with antioxidant N-acetyl cysteine (NAC) resulted in abrogation of EMT and stemness in HK-2 cells, indicating the role of nicotine-induced ROS in these morphological changes. The result also suggests that ROS controls the stemness through regulation of AKT pathway during early stages of carcinogenesis. Additionally, the expression of epigenetic regulatory genes was altered in nicotine-exposed cells and the changes were reversed by NAC. The epigenetic therapeutics 5-aza-2'-deoxycytidine and Trichostatin A also abrogated the stemness. This suggests the nicotine-induced oxidative stress caused epigenetic alterations contributing to stemness during neoplastic transformation. To our knowledge, this is the first report showing the ROS-mediated epigenetic modifications as the underlying mechanism for carcinogenicity of nicotine in human kidney epithelial cells. This study further suggests the potential of epigenetic therapeutics for pharmacological intervention in nicotine-induced kidney cancer.