Carrot is an important nutritional crop due to the high levels of pro-vitamin A carotenoids (β-carotene and, to a lower extent, α-carotene) that accumulate in its storage root during secondary growth. In this work we show that in carrots, contrary to that reported for aerial organs of other plant species, light has a profound effect on root development by inhibiting root thickening, preventing the differentiation of chromoplasts and eventually repressing the expression of most genes required for the biosynthesis of β-carotene and α-carotene and to a lesser extent genes for xanthophylls and apocarotenoids biosynthesis. We observed a correlation in the carotenoid profile and the patterns of gene expression during the development of root segments grown either in the light or in the dark, which suggests a transcriptional regulation for carotenoid synthesis during carrot root development. Furthermore, our work supports the conclusion that the differentiation of chromoplasts coincides with carotenoid accumulation during the later stages of development of underground storage roots.