【Objective】To evaluate the effects of the fecal status and transplantation method on intestinal flora structure after fecal microbiota transplantation in germ-free mice. 【Methods】Thirty-six C57BL/6 mice were randomly divided into three groups: fresh fecal gavage transplantation group, frozen fecal gavage transplantation group and frozen fecal rectal transplantation group. Feces were collected at 2 weeks, 4 weeks and 6 weeks after transplantation, and all mice were sacrificed at 6 weeks to obtain the contents of the small intestine and the large intestine. The structure and function of the gut microbiota and the dynamic trends of microbial changes were analyzed by 16S rRNA sequencing. 【Results】By α-diversity analysis, compared with the frozen fecal gavage transplantation group, the diversity of the small intestine microbes in both the fresh fecal gavage transplantation group and the frozen fecal rectal transplantation group showed no significant differences (P > 0.05), but the diversity of large intestine microbes was significantly increased (P < 0.001). By β-diversity analysis, samples from the small intestine of the fresh fecal gavage transplantation group and the frozen feces gavage transplantation group clustered in the same area, indicating that the microbial community composition was similar (P > 0.05). However, samples from the large intestine were distributed in different areas, showing significant differences (P = 0.001). For the frozen fecal gavage transplantation group and the frozen fecal rectal transplantation group, samples from both the small intestine and the large intestine were distributed in different areas, with significant differences (P = 0.001). By LEfSe analysis, Bacteroidota was relatively dominant in the fresh fecal gavage transplantation group, while Verrucomicrobiota and Proteobacteria were relatively dominant in the frozen fecal gavage transplantation group, and Firmicutes were relatively dominant in the frozen fecal rectal transplantation group. Functional prediction by PICRUSt2 showed that neither the fecal status nor the method of transplantation induced changes in the microbial community's functions. 【Conclusions】Compared to frozen fecal gavage transplantation, both fresh fecal gavage transplantation and frozen fecal rectal transplantation enhance the diversity of microbes in the large intestine. The fecal status does not affect the gut function and colonization trends of the microbiota, whereas the method of transplantation affects the colonization trends but not the functions of the microbiota.