Phytase (myo-inositol-1,2,3,4,5,6-hexakisphosphate phosphohydrolase, EC 3.1.3.26) catalyses the stepwise hydrolysis of phytic acid (myo-inositol hexakisphosphate). Phytases are of great commercial importance due to their usage as supplement of food and animal feed, which can cater to nutrition demands and alleviate environmental problems, has been approved by many countries. Although acid phytases have been extensively studied, information regarding the phytases from Citrobacter is limited. In the work presented, a phytase was separated from Citrobacter freundii. After steps of electrophoretic homogeneity by successive ammonium sulfate between 60% and 80% saturation precipitation, DEAE-Sepharose ion-exchange chromatography and gel filtration through Superdex HR 10/30, final gel elution resulted in a 41.3-fold purification and yield of 9.3%. Gel elution is an effective method to purify the protein which contaminated with a few other proteins. The purified preparations were used in subsequent characterization studies. Based on SDS-PAGE analysis, the molecular weight of the purified phytase was calculated to be approximately 45.0kDa in monomeric form. The pure enzyme has an optimum pH of 4.0 to approximately 4.5. It was found stable between pH5.0 to approximately 7.0, about 90% of the enzyme activity was retained at 37 degrees C for 60min. The phytase has an optimum temperature of 40 degrees C which was lower than that of other phytases from Aspergillus or E. coli (average 50 to approximately 60 degrees C) and was close to the temperature of gastrointestinal tract in animals (37 to approximately 40 degrees C). Thus the enzyme is a promising candidate for animal feed applications. Activity of the purified phytase was influenced by changing the reaction temperature. Data showed that the enzyme retained its activity over a long period when stored at 4 degrees C, whereas thermal inactivation studies indicated that the enzyme lost 100% activity after treatment at 60 degrees C for 4min. The Km values of the phytase for dodecasodium phytate at 37 degrees C was 0.85nmol/L with a Vmax 0.53IU/(mg x min). Phytase activity was strongly inhibited by SDS, Zn2+ and moderately inhibited by Cu2+, Cr3+, Fe2+ and Fe3+. Activity was not significantly affected by EDTA, K+, Mg2+ and Ca2+. The phytase has excellent resistance to trypsin, but not pepsin. The N-terminal amino acids sequence of the phytase protein was determined as QCAPEGYQLQQVLMM which exhibited about 80% homology to Glucose-1-phosphatases from E. coli, Shigella flexneri and Salmonella, whereas it did not show apparent sequence similarity with any other phytase listed in the databases. Initial characterization of the purified enzyme suggested that it is a potential candidate for use as an animal feed supplement.