Vitamin B6 is an essential coenzyme for numerous metabolic enzymes and is a potent antioxidant. In plants, very little is known about its contribution to viability, growth and development. The de novo pathway of vitamin B6 biosynthesis has only been described recently and involves the protein PDX1 (pyridoxal phosphate synthase protein). Arabidopsis thaliana has three homologs of PDX1, two of which, PDX1.1 and PDX1.3, have been demonstrated as functional in vitamin B6 biosynthesis in vitro and by yeast complementation. In this study, we show that the spatial and temporal expression patterns of PDX1.1 and PDX1.3, investigated at the transcript and protein level, largely overlap, but PDX1.3 is more abundant than PDX1.1. Development of single pdx1.1 and pdx1.3 mutants is partially affected, whereas disruption of both genes causes embryo lethality at the globular stage. Detailed examination of the single mutants, in addition to those that only have a single functional copy of either gene, indicates that although these genes are partially redundant in vitamin B6 synthesis, PDX1.3 is more requisite than PDX1.1. Developmental distinctions correlate with the vitamin B6 content. Furthermore, we provide evidence that in addition to being essential for plant growth and development, vitamin B6 also plays a role in stress tolerance and photoprotection of plants.