Carbohydrate response element-binding protein (ChREBP) is a basic helix-loop-helix/leucine zipper transcription factor that binds to the carbohydrate response element in the promoter of certain lipogenic and glycolytic genes. High glucose can activate ChREBP by releasing an intramolecular inhibition within the glucose-sensing module (GSM) that occurs in low glucose. We report here that the glucose response of GSM is mediated by cooperation between five conserved submodules known as Mondo conserved regions (MCRs) I through V within GSM. Deletion of individual MCRs leads to complete (for MCR II, III, and IV) or partial (MCR I) loss of glucose response of ChREBP. MCR IV is necessary and sufficient for inhibiting the transcriptional activity of ChREBP under low glucose. The roles of MCR II and III in glucose response of ChREBP are independent of and distinct from their function in controlling subcellular localization. We further demonstrate that, instead of inhibiting ChREBP activity as would be predicted from its cytoplasmic retentive function, 14-3-3 binding with MCR III is essential for the glucose responsiveness of ChREBP. The interaction between 14-3-3 and ChREBP is constitutive, indicating a permissive role of 14-3-3 in the glucose response of ChREBP. We further uncovered an unconventional 14-3-3 binding motif (residues 116–135) lacking phosphor-serine/threonine within MCR III, a predicted α-helix highly conserved in all Mondo proteins. We conclude that individual subdomains in the GSM (MCR I through V) play diverse but crucial roles in cooperation with essential trans-acting cofactors such as 14-3-3 proteins to mediate the glucose response of ChREBP.