Vitamin B6 is an important molecule for all organisms, being a cofactor in 140 enzymatic reactions in the form of pyridoxal-5'-phosphate (PLP). The de novo biosynthetic pathway operating in most organisms, involves two enzymes, PDX1 and PDX2, to produce directly PLP and its regulation was the focus of this study. We have overproduced vitamin B6 in Arabidopsis thaliana by overexpressing PDX1.1 gene alone or in combination with PDX2, resulting in enhanced resistance to abiotic stress and increased organ size. We have demonstrated that the non-catalytic PDX1.2 homolog is induced by singlet oxygen and heat stress in vivo increasing vitamin B6 levels and contributing to plant survival. We have identified some of the factors involved in the differential regulation of the PDX1.1 and PDX1.3 genes. Finally, we have shown that increased levels of vitamin B6 can decrease the impact of singlet oxygen mediated oxidative stress in vivo and improve plant performance.