Yersinia pestis is the causative agent of plague. Both cyclic AMP receptor protein (CRP) and PhoP are involved in regulating virulence-related genes in Y. pestis. The phoPQ loci are transcribed as two distinct operons, YPO1635-phoPQ-YPO1632 and phoPQ-YPO1632. In the present work, the regulation of the first operon by CRP was investigated using primer extension, LacZ fusion, electrophoretic mobility shift assay, and DNase I footprinting assays. The results showed that CRP bound to a DNA region overlapping core promoter -10 element and transcription start of YPO1635 to repress the expression of YPO1635-phoPQ-YPO1632. Taken together with our previous results, complex regulatory interactions of CRP-AMP and PhoP/PhoQ were proposed in Y. pestis, which would contribute to tightly controlled expression of virulence-related genes.