Background: Traditional thinking is that physical activity benefits mental and physical health, however, excessive physical activity can increase anxiety, depression, and affect the gut microbiome. Considering the strong connection between the gut and the brain, the purpose of the present study was to evaluate the association between gut microbiota composition and anxiety as well as depression in highly active individuals. Methods: Participants included 55 young adults (ages 18-25, 51% males). All participants were highly physically active, as determined by 7 days of SenseWear monitoring. Anxiety and depression were measured with the Beck Anxiety and Depression Inventories. Alpha diversity, beta diversity, and microbial composition were evaluated via 16S rRNA gene sequencing using distal gut samples. Results: Greater anxiety was associated with both lower distal gut alpha diversity ( P < 0.05) and higher beta diversity (PERMANOVA test; R-squared: 0.17562, P = 0.027), which appeared stronger in males. Genus level taxonomic abundance analysis showed Prevotella relative abundance as higher in males with higher anxiety ( P = 0.03, q=0.06). However, adjusted linear regression analysis, controlling for fiber intake and sex nullified the association between Prevotella and anxiety. Additional analysis demonstrated a strong association between lower dietary fiber intake and higher anxiety scores (Est.= -0.48, SE= 0.20 , P = 0.021). Conclusion: In highly active individuals, specifically males, there is a strong relationship between the gut microbiome, fiber intake, and anxiety. These data suggest highly active males with anxiety may benefit from increased dietary fiber intake.