Objective To evaluate the effects of fecal status and transplantation method on the intestinal flora structure after fecal microbiota transplantation in germ-free mice. Methods Thirty-six C57BL/6 mice were divided randomly into three groups: fresh fecal gavage transplantation (group A), frozen fecal gavage transplantation (group B), and frozen fecal rectal transplantation groups (group C). Feces were collected at 2, 4 and 6 weeks after transplantation. All mice were sacrificed at 6 weeks to obtain the contents of the small and large intestines. The structure and function of the gut microbiota and dynamic trends in microbial changes were analyzed by 16S rRNA sequencing. Results The diversity of the small intestine microbes in both the group A and group C were similar to those in the group B, according to α-diversity analysis (P>0.05), but the diversity of large intestine microbes was significantly increased (P<0.001). According to β-diversity analysis, small intestine samples from the group A and group B clustered in the same area, indicating that the microbial community compositions were similar (P>0.05), but samples from the large intestine were distributed in different areas, showing significant differences (P<0.001). Small and large intestine samples from the group B and group C were distributed in different areas, with significant differences (P<0.001). Linear discriminant analysis effect size showed that Bacteroidota were relatively dominant in the group A, while Verrucomicrobiota and Proteobacteria were relatively dominant in the group B and Firmicutes were relatively dominant in the group C. Functional prediction using PICRUSt2 software showed that neither fecal status nor the method of transplantation affected the functions of the microbial community. Conclusions Both fresh fecal gavage and frozen fecal rectal transplantation can enhance the microbial diversity of the large intestine, compared with frozen fecal gavage transplantation. 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.