["Journal of Cellular Physiology, Volume 241, Issue 6, June 2026. ", "This study identifies S100A4 as a critical regulator of NMIIA assembly, phosphorylation, and actin cytoskeletal organization in the ocular lens. Loss of S100A4 disrupts lens fiber cell architecture and impairs lens transparency, revealing a previously unrecognized role for S100A4 in maintaining lens homeostasis and structural integrity.\n\n\n\n\n\nABSTRACT\nNon‐muscle myosin IIA (NMIIA), a motor protein plays a critical role in regulating cell morphology, adhesion, migration, contractility, and mechanotransduction across various tissues, including the ocular lens. S100A4, a known NMIIA‐interacting protein, is abundantly expressed and exhibits a discrete spatial distribution in lens fibers. Loss of S100A4 has previously been shown to associate with late‐onset lens opacification in mice. However, its role in regulating NMIIA activity, assembly, and actin cytoskeletal organization in the lens remains unclear. Using S100A4‐null mice, this study reveals that S100A4 co‐immunoprecipitates with NMIIA and that its absence leads to decreased NMIIA (Ser1943) phosphorylation, impaired NMIIA filament assembly, and disruption in actin cytoskeletal organization and polymerization in the lens. Quantitative proteomic analysis further identified decreased levels of CLIC5 and RNA binding protein‐SERBP1 in cytoskeletal‐ and membrane‐enriched fractions from S100A4‐null versus wild‐type lenses. Moreover, treatment of wild‐type mouse lenses in ex‐vivo with trifluoperazine, a known S100A4 inhibitor, induced lens opacification in association with increased insolubilization of S100A4, NMIIA and actin. Collectively, these findings demonstrate that S100A4 plays a critical role in regulating NMIIA activity and assembly, and maintaining actin cytoskeletal organization in the ocular lens, thereby contributing to lens transparency and homeostasis."]