Alcoholic fermentation is a crucial step of winemaking, during which yeasts convert sugars to alcohol and also produce or biotransform numerous flavour compounds. In this context, nutrients are essential compounds to support yeast growth and ultimately ensure complete fermentation, as well as optimized production of flavour compounds over that of off-flavour compounds. In particular, the vitamin thiamine not only plays an essential cofactor role for several enzymes involved in various metabolic pathways, including those leading to the production of wine-relevant flavour compounds, but also aids yeast survival via thiamine-dependent stress protection functions. Most yeast species are able to both assimilate exogenous thiamine into the cell and synthesize thiamine de novo. However, the mechanism and level of thiamine accumulation depend on several factors. This review provides an in-depth overview of thiamine utilization and metabolism in the model yeast species Saccharomyces cerevisiae, as well as the current knowledge on (1) the intracellular functions of thiamine, (2) the balance between and regulation of uptake and synthesis of thiamine and (3) the multitude of factors influencing thiamine availability and utilization. For the latter, a particular emphasis is placed on conditions occurring during wine fermentation. The adequacy of thiamine concentration in grape must to ensure successful fermentation is discussed together with the effect of thiamine concentration on fermentation kinetics and on wine sensory properties. This knowledge may serve as a resource to optimise thiamine concentrations for optimal industrial application of yeasts.