Rice is a paramount food crop to a large portion of the human population. Unfortunately, the starchy endosperm, which is the remaining portion of the seed following polishing practices, contains limited amounts of micronutrients. Here, a metabolic engineering approach is conducted, aimed at enhancing the level of thiamin (vitamin B1), specifically in the endosperm. To achieve this, major thiamin biosynthesis genes, controlled by strong endosperm specific promoters, were introduced in the engineered rice lines. Three different approaches were explored, using different gene combinations. Determination of thiamin and thiamin biosynthesis intermediates reveals the importance of full pathway engineering to achieve adequate flux through the biosynthesis pathway of this vitamin. Up to fourfold higher thiamin levels as compared to WT were reached. These results confirm the ability of metabolic engineering approaches to enhance de novo thiamin biosynthesis in rice endosperm tissue.