Alkaloids are important compounds found in Nicotiana plants, essential in plant defense against herbivores. The main alkaloid of Nicotiana tabacum, nicotine, is produced in roots and translocated to the leaves. Nicotine is formed by a pyrrolidine and a pyridine ring in a process involving several enzymes. The pyridine ring of nicotine is derived from nicotinic acid, whereas the pyrrolidine ring originates from polyamine putrescine metabolism. After synthesis in root cortical cells, a set of transporters is known to transport nicotine upward to the aerial part and store it in leaf vacuoles. Moreover, nicotine can be metabolized in leaves, giving rise to nornicotine through the N-demethylation process. Some Nicotiana wild species produce acyltransferase enzymes, which allow the plant to make N-acyl-nornicotine, an alkaloid with more potent insecticidal properties than nicotine. However, although we can find a wealth of information about the alkaloid production in Nicotiana spp., our understanding about nicotine biosynthesis, transport, and metabolism is still incomplete. This review will summarize these pathways on the basis on recent literature, as well as highlighting questions that need further investigation.