Micronutrient deficiency affects an estimated 2 billion people worldwide. Vitamin B1 is an essential micronutrient, and inadequate intake can result in certain deficiency disorders. Rice, a staple crop for 50% of the world's population, is a poor source of several micronutrients, when polished rice is the chief source of calories in the absence of dietary diversity. This study aimed to biofortify rice endosperm with enhanced levels of vitamin B1. Strategies were devised to explore the potential of biosynthesis de novo, sequestration and transport of vitamin B1 in this context. Notably, as potential vitamin B1 cotransporters, the POLYAMINE UPTAKE TRANSPORTER (PUT) family were observed to be disturbed in polyamine metabolism and had associated developmental phenotypes. However, it was shown that they are unlikely to be involved in vitamin B1 transport in rice. A sequestration strategy for vitamin B1 with a putative binding protein retains potential for future investigation.