Tobacco consumption increases the susceptibility to develop infectious diseases such as tuberculosis (TB). Nicotine (Nc) is the main component of cigarette smoke with immunomodulatory properties, however, its effect on Mycobacterium tuberculosis (Mtb) has been scarcely investigated. The present study evaluated the effect of nicotine on the growth of Mtb and on the induction of virulence-related genes. Mycobacteria were exposed to different concentrations of nicotine then Mtb growth was evaluated. Subsequently, the expression of the virulence-related genes lysX, pirG, fad26, fbpa, ompa, hbhA, esxA, esxB, hspx, katG, lpqh, and caeA was evaluated by RT-qPCR. The effect of nicotine on intracellular Mtb was also evaluated. The results showed that nicotine promotes the growth of Mtb both extracellularly and intracellularly and increases the expression of genes related to virulence. In summary, nicotine promotes the growth of Mtb and the expression of virulence-related genes that could be correlated with the increased the risk of smokers developing TB.