<p>Trail development is more prevalent as tourism develops globally. The depth effect of trail development on plant diversity and native species’ stress response via tuning flavonoids in natural ecosystems remain relatively poorly understood. We investigated the depth effects by comparing plant species diversity and flavonoid contents (of six common native species) in sampling plots plots (Rabbit Mountain Open Space, Boulder County, CO, USA) with varying distances away from trail. We found plant diversity to be lowest in plots immediately proximal to trails and highest in intermediate plots. We also found the concentrations of total flavonoids to vary significantly between plots closer and away from trails. Specifically, we found the concentrations of isoorientin and myricetin higher in plots closer to trails. On the contrary, the concentrations of vitexin and kaempferol were higher in plots away from trails. Quercetin was higher in the intermediate plots. Overall, trail development negatively impacted herbaceous plant diversity, which was evident as depth effects. The plant species responded to environmental stresses imposed by trail development through fine-tuned flavonoid accumulation.</p>