Bacteria residing in the human gastrointestinal tract has a symbiotic relationship with its host. Animal models have demonstrated a relationship between exercise and gut microbiota composition. This was the first study to explore the relationship between cardiorespiratory fitness (maximal oxygen consumption, VO2max) and relative gut microbiota composition (Firmicutes to Bacteroidetes ratio [F/B]) in healthy young adults in a free-living environment. Twenty males and 17 females (25.7 ± 2.2 years), who did not take antibiotics in the last 6 months, volunteered for this study. VO2max was measured using a symptom-limited graded treadmill test. Relative microbiota composition was determined by analyzing DNA extracted from stool samples using a quantitative polymerase chain reaction that specifically measured the quantity of a target gene (16S rRNA) found in Firmicutes and Bacteroidetes. Relationships between F/B and potentially related dietary, anthropometric, and fitness variables were assessed using correlation analyses with an appropriate Bonferroni adjustment (p < .004). The average F/B ratio in all participants was 0.94 ± 0.03. The F/B ratio was significantly correlated to VO2max (r = .48, p < .003), but no other fitness, nutritional intake, or anthropometric variables (p > .004). VO2max explained ∼22% of the variance of an individual’s relative gut bacteria as determined by the F/B ratio. These data support animal findings, demonstrating a relationship between relative human gut microbiota composition and cardiorespiratory fitness in healthy young adults. Gastrointestinal bacteria is integral in regulating a myriad of physiological processes, and greater insight regarding ramifications of exercise and nutrition on gut microbial composition may help guide therapies to promote human health.