Cigarette smoking is strongly correlated with the onset of lung cancer. Nicotine, a major component in cigarette smoke, has been found to promote tumor growth and angiogenesis, as well as protect cancer cells from apoptosis. Among all lung cancer cases, small cell lung cancer (SCLC) is found almost exclusively in smokers; metastasis and chemoresistance are the main reasons for the high mortality rates associated with SCLC. Retrospective studies have shown that patients with tobacco-related cancers who continue to smoke after their diagnosis display lower response rates and a shorter median survival compared with those who stop smoking. In the current work, we examined the effects of acute and repetitive exposure to nicotine, in the concentrations found in the lungs of active smokers, on the malignant properties of N417 SCLC cells in vitro. We observed that repetitive nicotine exposure induced a neuronal-like appearance in N417 cells along with increased adhesion to the extracellular matrix and chemoresistance. These changes were accompanied by enhanced migration through collagen matrices and adhesion to and transmigration across lymphatic endothelial cell monolayers. SCLC differentiation reverted after cessation of nicotine exposure. Here, we provide evidence for the leading role of the CXCR4/CXCL12 axis in these phenomena. Finally, we show how nicotine-differentiated N417 cells produced bigger and more vascularized tumors in mice, with lower apoptotic rates, than their nondifferentiated counterparts. In short, these findings identify the mechanisms through which nicotine increases SCLC malignancy and provide further evidence that CXCR4 is a potential anticancer target for nicotine-associated SCLC.