Epimedium species, valued for bioactive flavonoids like icariin and epimedins, face cultivation challenges due to heterogeneous soil conditions in karst ecosystems. This study examined how key soil properties (bulk density, porosity, nutrients) regulate flavonoid accumulation across wild, open-field, and understory cultivation systems in Guizhou Province, China, aiming to guide precision agroecology for enhanced medicinal quality. Samples from three cultivation systems in Qiandongnan and Tongren were analyzed for soil parameters (density, porosity, NPK) and flavonoid content via HPLC-UV. Multivariate analyses (PCA, RDA) quantified soil-phytochemical interactions, validated by permutation tests. Soil heterogeneity significantly influenced flavonoid levels: wild (4.37% ±0.52), open-field (10.19% ±1.21), and understory (8.95% ±0.89). PCA differentiated cultivation systems (65.3% variance), while RDA identified soil organic matter (p<0.001) and hydraulic conductivity (p<0.01) as primary drivers of flavonoid variation. Strategic soil management—optimizing organic matter (>3.2%) and reducing compaction (<1.4 g/cm 3)—can elevate Epimedium medicinal quality by 28–41%. These findings provide actionable insights for sustainable cultivation, aligning ecological adaptability with industrial standardization in karst regions.