BACKGROUND: Thioredoxin interacting protein (Txnip) is a multifunctional protein involved in regulation of cell cycle events and cellular metabolism. The expression of Txnip is known to be induced by glucose, adenosine-containing molecules, and other physiological cues; however, the underlying regulatory mechanisms remain elusive. METHODOLOGY/PRINCIPAL FINDINGS: In this study, using promoter reporter, electrophoresis mobility shift (EMSA), and chromatin immuno-precipitation (ChIP) assays, we have identified an additional carbohydrate response element (ChoRE) on the promoter of Txnip gene, which functions cooperatively with the earlier identified ChoRE to mediate optimal Txnip expression. However, these two ChoREs are not sufficient to mediate the induction of Txnip expression by glucose or adenosine-containing molecules; and two CCAAT boxes, both of which can recruit nuclear factor Y (NF-Y) to the Txnip promoter, are also required for the induction. Accordingly, we have found that the function of ChoREs and associated factors is contingent on tandem CCAAT boxes, in that occupancy of the Txnip promoter by NF-Y is a prerequisite for efficacious recruitment of Mondo/MLX to ChoREs under glucose stimulation. CONCLUSIONS/SIGNIFICANCE: Our findings suggest a synergy between the tandem CCAAT and ChoRE motifs and associated NF-Y and Mondo/MLX transcription factors in enhancing transcription from the Txnip promoter. This piece of information will be helpful for future dissection of molecular mechanisms governing the transcriptional regulation of Txnip, a glucose responsive gene.