As a versatile cofactor for many enzymes catalyzing important biochemical reactions, vitamin B6 is required for all cellular organisms. In contrast to bacteria, fungi and plants, which have the ability to synthesize vitamin B6de novo, animals have to take up the vitamin from their diet. Plants are the major source of vitamin B6 for animals. The recent identification of vitamin B6 biosynthetic enzymes PDX1 and PDX2 in plants makes it possible to regulate the biosynthesis of this important vitamin. In this study, we generated Arabidopsis plants overexpressing the PDX1 and/or PDX2 gene and used a liquid chromatography/mass spectrometry/mass spectrometry method to determine the levels of different forms of vitamin B6 in these transgenic plants. It was found that expression of the PDX genes under control of the CaMV 35S promoter caused only a limited increase in pyridoxine contents in dry seeds but not in shoots or roots. When using the Arabidopsis seed-specific 12S promoter to drive the expression of the PDX genes, the levels of vitamin B6 increased more than twofold in transgenic plants. Our work demonstrates that it is feasible to enhance vitamin B6 content in seeds by metabolic engineering.