Chronic kidney fibrosis poses a significant global health challenge with effective therapeutic strategies remaining elusive. While cell-extracellular matrix (ECM) interactions are known to drive fibrosis progression, the specific role of focal adhesions (FAs) in kidney fibrosis is not fully understood. In this study, we investigated the role of FAs in kidney tubular epithelial cell fibrosis by employing precise nanogold patterning to modulate integrin distribution. We demonstrate that increasing ligand spacing disrupts integrin clustering, thereby inhibiting FA formation and attenuating fibrosis. Importantly, enhanced FA activity is associated with kidney fibrosis in both human disease specimens and murine models. Mechanistically, FAs regulate fibrosis through mechanotransduction pathways, and our in vivo experiments show that suppressing mechanotransduction significantly mitigates kidney fibrosis in mice. These findings highlight the potential of targeting FAs as a therapeutic strategy, offering new insights into clinical intervention in kidney fibrosis.