Vitamin B₆(pyridoxine) is vital for key metabolic reactions and reported to have antioxidant properties in planta. Therefore, enhancement of vitamin B₆content has been hypothesized to be a route to improve resistance to biotic and abiotic stresses. Most of the current studies on vitamin B₆in plants are on eudicot species, with monocots remaining largely unexplored. In this study, we investigated vitamin B₆biosynthesis in rice, with a view to examining the feasibility and impact of enhancing vitamin B₆levels. Constitutive expression in rice of two Arabidopsis thaliana genes from the vitamin B₆biosynthesis de novo pathway, AtPDX1.1 and AtPDX2, resulted in a considerable increase in vitamin B₆in leaves (up to 28.3-fold) and roots (up to 12-fold), with minimal impact on general growth. Rice lines accumulating high levels of vitamin B₆did not display enhanced tolerance to abiotic stress (salt) or biotic stress (resistance to Xanthomonas oryzae infection). While a significant increase in vitamin B₆content could also be achieved in rice seeds (up to 3.1-fold), the increase was largely due to its accumulation in seed coat and embryo tissues, with little enhancement observed in the endosperm. However, seed yield was affected in some vitamin B₆-enhanced lines. Notably, expression of the transgenes did not affect the expression of the endogenous rice PDX genes. Intriguingly, despite transgene expression in leaves and seeds, the corresponding proteins were only detectable in leaves and could not be observed in seeds, possibly pointing to a mode of regulation in this organ.