Objective To investigate the association of physical fitness levels with gut microbiota and find out the bacterial taxa with potential of improving physical fitness. Methods 44 healthy adult men were recruited for this study and the data on physical fitness was obtained. They were assigned to a high or low physical fitness group by using latent profile analysis. High⁃throughput sequencing was used to detect 16S rDNA collected from the fecal samples of volunteers before physical test. The alpha or beta diversity of intestinal flora was analyzed to deter⁃ mine the differences in gut flora between the two groups. Linear discriminant analysis Effect Size was applied to screen the taxa contributing more to the diversity，which were considered as closely related to physical fitness level. Spearman correlation analysis was used to determine the correlation among the richness of taxa that contribute more to diversity and indexes related to physical fitness. Results There wahs no significant difference in the alpha diversity of gut flora between the two groups，whereas the beta diversity differed significantly. Lachnospiraceae ND3007 group and Clostridia UCG⁃014 were related to higher level of physical fitness，while Olsenella，Bacteroi⁃ des stercoris and Eubacterium ventriosum group were associated with lower lever of physical fitness. The richness of taxa related to better physical fitness condition appeared to be negatively correlated with that of worse physical fitness condition，and the latter showed a stronger correlation with indexes related to physical fitness than the former. Conclusions The composition of microbiota planted in gastrointestinal tract was associated with physical fitness. Lachnospiraceae ND3007 group could be considered as latent taxa to help improve physical fitness condition.