Tocopherols, with antioxidant properties, are synthesized by photosynthetic organisms and play important roles in human and animal nutrition. In soybean, γ-tocopherol, the biosynthetic precursor to α-tocopherol, is the predominant form found in the seed, whereas α-tocopherol is the most bioactive component. This suggests that the final step of the α-tocopherol biosynthetic pathway catalyzed by γ-tocopherol methyltransferase (γ-TMT) is limiting in soybean seed. Soybean oil is the major edible vegetable oil consumed, so manipulating the tocopherol biosynthetic pathway in soybean seed to convert tocopherols into more active α-tocopherol form could have significant health benefits. In order to increase the soybean seed α-tocopherol content, the γ-TMT gene isolated from Perilla frutescens was overexpressed in soybean using a seed-specific promoter. One transgenic plant was recovered and the progeny was analyzed for two generations. Our results demonstrated that the seed-specific expression of the P. frutescens γ-TMT gene resulted in a 10.4-fold increase in the α-tocopherol content and a 14.9-fold increase in the β-tocopherol content in T2 seed. Given the relative contributions of different tocopherols to vitamin E activity, the activity in T2 seed was calculated to be 4.8-fold higher than in wild-type seed. In addition, the data obtained on lipid peroxidation indicates that α-tocopherol may have a role in preventing oxidative damage to lipid components during seed storage and seed germination. The increase in the α-tocopherol content in the soybean seed could have a potential to significantly increase the dietary intake of vitamin E.