Background <p>Melanoma, a highly metastatic and treatment-resistant malignancy, urgently requires novel therapeutic strategies. Puerarin, a natural isoflavone with established anti-tumorigenic effects in diverse cancers, remains underexplored in melanoma despite its potential to modulate melanogenesis and oxidative stress. This study investigates puerarin’s spatial targeting mechanisms in melanoma to elucidate its therapeutic specificity.</p> Methods <p>A multi-omics approach integrating single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, and structure-based docking plus normal-mode analysis was employed. Spatial domain analysis identified puerarin-responsive malignant cell clusters, while molecular docking and protein-ligand simulations prioritized CD44 as a candidate receptor. Functional validation included extracellular matrix (ECM) signaling pathway analysis, spatial colocalization studies, and in vitro experiments.</p> Results <p>Spatial analysis showed enrichment of puerarin-related signals in malignant-cell–dominated domains. ECM ligands (collagens, fibronectin, laminins) that signal through CD44/SDC1 were concentrated in these domains, where CD44 was overexpressed relative to surrounding compartments. Molecular modeling suggested CD44, a cell-surface receptor overexpressed in melanoma cells, as a putative mediator of puerarin’s effects on these pathways. In vitro experiments further supported CD44’s role in modulating puerarin-responsive domains.</p> Conclusion <p>These observations are hypothesis-generating and provide a potential direction for future research on whether CD44 mediates puerarin’s spatial effects in melanoma.</p>