Plants synthesize l-ascorbic acid (AsA) by the Smirnoff-Wheeler pathway, which is considered to be the principal de novo biosynthesis pathway for AsA. In addition, three alternative biosynthesis pathways have also been proposed in plants. The AsA levels are different among organs, and they are also affected by internal factors such as growth and development as well as the environmental factors such as light, drought, salt, and extreme temperature. Among them, ascorbic acid biosynthesis in plants is mostly stimulated by light, depending on its quantity. The AsA levels are well regulated at the transcriptional and posttranscriptional levels of AsA biosynthetic enzymes. The AsA levels are considerably different among plant species. Acerola (Malpighia glabra), a tropical plant with a high levels of AsA in its fruits, is a powerful tool for revealing how to regulate and increase AsA levels in plants. Gene expression analysis in acerola has allowed authors to propose that high transcriptional expression level of the biosynthetic enzymes may contribute to high accumulation of AsA. The upstream regions of the genes involved in AsA biosynthesis contain the cis-element(s) required for specificity and high level of their gene expression. Because of its antioxidant role and nutrient value, one of the goals to understand the regulation of AsA biosynthesis is to manipulate AsA levels in plants. This chapter focuses on expression and regulation of genes involved in AsA biosynthesis of higher plants.