The present invention can grow in tumor tissues under anaerobic environment, it is possible to express the cytosine deaminase (CD), and at least anti-tumor activity effective concentration of 5-fluorouracil (5-FU) resisting performance Preparation method of CD expression · 5-FU-resistant bacterium having there is provided a. Specifically, the CD expression bacteria can grow in tumor tissues under anaerobic environment, a method A passaging-conditioned culture in the presence of 5-fluorocytosine (5-FC), (1) anaerobic can grow in tumor tissue is under environmental and a step of passaged-conditioned culture the bacteria do not express CD in the presence of 5-FU, to produce a 5-FU-resistant bacteria, (2) the 5 the -FU resistant bacteria, a method B and a step of transforming by introducing CD gene. The present invention is to provide a pharmaceutical composition containing the CD expression · 5-FU-resistant bacteria and this.