Histidine phosphorylation is an underexplored form of protein phosphorylation. Despite the widespread existence of phosphohistidine (pHis) sites, particularly in bacteria, their phosphorylation regulation and physiological functions remain poorly understood. In this study, we developed a chemoproteomic strategy employing a stable pHis analog to identify pHis-recognizing proteins in Escherichia coli. Our probe successfully labeled known pHis-recognizers and revealed many putative pHis acceptors, including phosphofructokinase-1 (PfkA), a key glycolytic enzyme. We demonstrated that PfkA undergoes histidine phosphorylation at His249 mediated by the phosphocarrier protein PtsH, thereby reducing enzyme activity. This phosphorylation was reversed by the pHis-specific phosphatase SixA, which restored the PfkA activity. Our findings reveal a novel posttranslational regulatory mechanism affecting glycolysis, implicating a broader role of histidine phosphorylation in bacterial metabolic control.