The biosynthesis of histidine, a proteinogenic amino acid, has been extensively studied due to its importance in bacterial growth and survival. Histidinol-phosphate phosphatase (Hol-Pase), which is responsible for the penultimate step of histidine biosynthesis, is generally the last enzyme to be characterized in many bacteria because its origin and evolution are more complex compared to other enzymes in histidine biosynthesis. However, none of the enzymes in histidine biosynthesis, including Hol-Pase, have been characterized in , which is an important opportunistic Gram-negative pathogen that can cause serious human infections. In our previous work, a transposon mutant of was found to display a growth defect on glucose-containing minimal solid medium. In this study, we found that the growth defect was due to incomplete histidine auxotrophy caused by PA0335 inactivation. Subsequently, PA0335 was shown to encode Hol-Pase, and its function and enzymatic activity were investigated using genetic and biochemical methods. In addition to PA0335, the roles of 12 other predicted genes involved in histidine biosynthesis in were examined. Among them, (PA3165), (PA3152), and (PA3151) were found to be dispensable for histidine synthesis, whereas (PA4449), (PA5067), (PA5140), (PA5143), (PA5066), (PA4447), and (PA5141) were essential because deletion of each resulted in complete histidine auxotrophy; similar to the case for PA0335, (PA5142) or (PA4448) deletion caused incomplete histidine auxotrophy. Taken together, our results outline the histidine synthesis pathway of Histidine is a common amino acid in proteins. Because it plays critical roles in bacterial metabolism, its biosynthetic pathway in many bacteria has been elucidated. However, the pathway remains unclear in , an important opportunistic pathogen in clinical settings; in particular, there is scant knowledge about histidinol-phosphate phosphatase (Hol-Pase), which has a complex origin and evolution. In this study, Hol-Pase was identified and characterized. Furthermore, the roles of all other predicted genes involved in histidine biosynthesis were examined. Our results illustrate the histidine synthesis pathway of The knowledge obtained from this study may help in developing strategies to control -related infections. In addition, some enzymes of the histidine synthesis pathway from might be used as elements of histidine synthetic biology in other industrial microorganisms.