Background: Thiamine availability is involved in glycolytic flux and fermentation efficiency. A deficiency of this vitamin may be responsible for sluggish fermentations in wine making. Therefore, both thiamine uptake and de novo synthesis could have key roles in fermentation processes. Thiamine biosynthesis is regulated in response to thiamine availability and is coordinated by the thiamine sensor Thi3p, which activates Pdc2p and Thi2p. We used a genetic approach to identify quantitative trait loci (QTLs) in wine yeast and we discovered that a set of thiamine genes displayed expression-QTL on a common locus, which contains the thiamine regulator THI3. Results: We deciphered here the source of these regulatory variations of the THI and PDC genes. We showed that alteration of THI3 results in reduced expression of the genes involved in thiamine biosynthesis (THI11/12/13 and THI74) and increased expression of the pyruvate decarboxylase gene PDC1. Functional analysis of the allelic effect of THI3 confirmed the control of the THI and PDC1 genes. We observed, however, only a small effect of the THI3 on fermentation kinetics. We demonstrated that the expression levels of several THI genes are correlated with fermentation rate, suggesting that decarboxylation activity could drive gene expression through a modulation of thiamine content. Our data also reveals a new role of Thi3p in the regulation of the main pyruvate decarboxylase gene, PDC1. Conclusions: This highlights a switch from PDC1 to PDC5 gene expression during thiamine deficiency, which may improve the thiamine affinity or conservation during the enzymatic reaction. In addition, we observed that the lab allele of THI3 and of the thiamin transporter THI7 have diverged from the original alleles, consistent with an adaptation of lab strains to rich media containing an excess of thiamine.