Tocopherols (vitamin E) play essential roles in human health due to their antioxidant activity, and plant-derived oils are the richest sources of tocopherols in the human diet. Although soybean (Glycine max) is one of the main sources of plant-derived oil and tocopherol in the world, the relationship between tocopherol and oil in soybean seeds remains unclear. Here we focus on dissecting tocopherol metabolism, with the long-term goal of increasing α-tocopherol content and soybean oil quality. We first collected the tocopherol and fatty acid profiles in the soybean population (> 800 soybean accessions) and found that tocopherol content increased during soybean domestication. Meanwhile, a strong positive correlation between tocopherol and oil content was detected. Five tocopherol pathway-related loci were identified by using the metabolite genome-wide association study (mGWAS) strategy. Genetic variations of three tocopherol pathway genes are responsible for total-tocopherol and composition in soybean population, at either enzymatic activity level, mainly caused by nonconserved amino acid replacement or gene transcriptional level. Moreover, the fatty acid regulatory transcription factor GmZF351 directly activated tocopherol pathway gene expression and then increased both fatty acids and tocopherol in soybean seeds. Our study not only elucidates the functional differentiation of tocopherol pathway genes in soybean populations but also provides a feasible framework for developing new soybean varieties with high content of α-T and oil quality in seeds.