In previous studies, we have shown that RNA levels of the thiamine transporter THTR2 were down-regulated in breast cancer tumors in comparison with normal tissues and that THTR2-mediated increases in thiamine uptake activity contributed to increased apoptosis after exposure to ionizing radiation. To further understand the biological effects of the alteration of THTR2 expression, we conducted a DNA microarray study of gene expression in THTR2-transfected breast cancer cells and found that, in addition to increased expression of THTR2 attributable to the transgene, three other genes were up-regulated >2.5-fold in the transfected cells: cytochrome P450 isoform CYP4B1, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), and transcription factor CRIP1. In addition, two genes were confirmed to be down-regulated in THTR2-transfected cells: trefoil factor 1 (TFF1) and Rho-GDP dissociation inhibitor (RGDI). Up-regulation of 15-PGDH and CYP4B1 expression was observed in other breast cancer cell lines transfected with THTR2, and down-regulation was observed after suppression of THTR2 with siRNA vectors. To determine the role of exogenous thiamine in the expression of these genes, we analyzed THTR2-transfected breast cancer cells grown in thiamine-depleted medium by quantitative reverse transcription-PCR and showed that three of these five genes showed evidence of regulation by exogenous thiamine in a manner concordant with the effects of THTR2 overexpression. One of the genes up-regulated by THTR2 transfection was down-regulated by thiamine depletion (CYP4B1), and two genes with decreased expression in THTR2-transfected breast cancer cells were up-regulated by thiamine depletion (TFF1 and RGDI). In summary, these studies show unexpected relationships between thiamine metabolism and genes that may be involved in the oncogenesis of breast and lung cancer.