The aim of this study was to describe the expression and distribution of 14-3-3 zeta in trabecular meshwork (TM) cells and its regulatory role in the actomyosin system.The expression of 14-3-3 zeta was detected using Western blot analysis, RT-PCR, and immunofluorescence staining. TGF-β1 was used to induce cell contraction. Changes in the levels of 14-3-3 zeta, total RhoA, and the phosphorylation of myosin light chain (MLC) and cofilin were determined using Western blot analysis. The effects of 14-3-3 zeta knockdown on the actin cytoskeleton and focal adhesion were determined using immunofluorescence. The mRNA levels of fibronectin and collagen I and III were examined using quantitative RT-PCR. The contraction of TM cells was detected using collagen gel contraction (CGC) assays. The activation of the RhoA pathway was analyzed using a specific kit.The 14-3-3 zeta protein was highly expressed in TM cells. Down-regulation of 14-3-3 zeta resulted in the following: a decrease in the phosphorylation of both MLC and cofilin, a decrease in the formation of stress fibers and focal adhesion, alteration of the mRNA composition of the extracellular matrix (ECM), and the inhibition of TGF-β1-induced cell contraction. In addition, silencing of 14-3-3 zeta directly decreased total RhoA levels in TM cells.Collectively, our data suggest that 14-3-3 zeta plays a crucial role in regulating cytoskeletal structures, ECM homeostasis, and TGF-β1-induced contraction in TM cells by acting through the RhoA signaling pathway.