Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infection (UTI), a common bacterial infectious disease. This bacterium invades the urinary tract cells, where it aggregates, and subsequently forms multicellular colonies termed intracellular bacterial communities (IBCs). The motility of the bacteria plays a key role in the mechanism of virulence in the host bladder. Here, we show that CytR is a modulator of bacterial internalization and aggregation within the bladder epithelial cells sustained by CRP in UPEC. Mutational analyses and gel-shift assays indicated that CytR represses the expression of flhD, thereby encoding a master regulator for flagellar expression that is responsible for bacterial motility when CRP is present, whereas CRP is an activator of flhD expression. Thus, elevated flagellar expression was involved in promoted virulence in the cytR mutant. These combined observations suggest another regulatory layer of flagellar expression and the role of CytR in UPEC virulence. (C) 2019 Elsevier Inc. All rights reserved.