Several epidemiological studies have demonstrated that high vitamin E intakes are related to a reduced risk of non-communicable diseases, while other dietary antioxidants are not, suggesting that vitamin E exerts specific healthy functions in addition to its antioxidant role. In this regard, tomato (Solanum lycopersicum), one of the most consumed vegetables of the whole world population, is an important source of both tocopherols and tocotrienols. However, vitamin E content may strongly depend on several biotic and abiotic factors. In this review we will debate the elements affecting the synthesis of tocopherols and tocotrienols in tomato fruit, such as environmental conditions, genotype, fruit maturity level, and the impact of classical processing methods, such as pasteurization and lyophilization on the amount of these compounds. In addition we will analyze the specific vitamin E mechanisms of action in humans and the consequent functional effects derived from its dietary intake. Finally, we will examine the currently available molecular techniques used to increase the content of vitamin E in tomato fruit, starting from the identification of genetic determinants and quantitative trait loci that control the accumulation of these metabolites.