The cAMP receptor protein (CRP) coordinates carbon-responsive gene expression in Escherichia coli , but its direct role in trehalose biosynthesis has remained unclear. This study investigated whether CRP directly regulates the otsBA locus through the otsB promoter. RT–qPCR showed that otsB and otsA expression was strongly increased in a Δ crp mutant and under cAMP-deficient conditions. EMSA demonstrated direct association of CRP with the otsB promoter in the presence of cAMP, whereas trace-level DNase I reanalysis identified candidate CRP-associated protection windows near the promoter. Promoter-reporter assays and chromosomal promoter mutagenesis showed that the predicted promoter element is required for CRP-dependent repression under the tested conditions. Disruption of this element was associated with an altered relative intracellular carbohydrate profile and a modest growth difference under the tested hyperosmotic condition. CRP directly represses the otsB promoter, thereby negatively regulating the otsBA locus via a promoter-proximal regulatory element. These findings connect carbon-responsive transcriptional control with trehalose biosynthesis and osmotic stress-associated physiology in E. coli .