The human thiamine transporter-1 (hTHTR-1) contributes to intestinal thiamine uptake, and its function is regulated at both the transcriptional and posttranscriptional levels. Nothing, however, is known about the protein(s) that may interact with hTHTR-1 and affects its cell biology and physiology. We addressed this issue in the present investigation using a bacterial two-hybrid system to screen a human intestinal cDNA library with the complete coding sequence of hTHTR-1 as a bait. Our results showed that a member of the tetraspanin family of proteins, Tspan-1, interacts with hTHTR-1. Coimmunoprecipitation and glutathione S-transferase (GST)-pulldown assays confirmed the existence of such an interaction between hTspan-1 and hTHTR-1 in human intestinal epithelial Caco-2 cells. Furthermore, live cell confocal imaging demonstrated that hTspan-1 and hTHTR-1 colocalize in human intestinal epithelial HuTu-80 cells. The importance of the interaction between hTspan-1 and hTHTR-1 for cell biology of the thiamine transporter was examined in HuTu-80 cells stably expressing hTHTR-1. Coexpression of hTspan-1 in these cells led to a significant decrease in the rate of degradation of hTHTR-1 compared with cells expressing the hTHTR-1 alone; in fact the half-life of the hTHTR-1 protein was twice longer in the former cell type compared with the latter cell type (12 h vs. 6 h, respectively). This finding was also confirmed at the functional level when a significantly higher thiamine uptake was observed in cycloheximide-treated (6 h) cells expressing hTHTR-1 together with hTspan-1 compared with those expressing hTHTR-1 alone. These studies demonstrate for the first time that Tspan-1 is an interacting partner with hTHTR-1 and that this interaction affects hTHTR-1 stability.