AIM: To research the effect of Y-27632, a selective Rho-associated coiled-coil kinase (ROCK) inhibitor, on TGF-β1/Smad2, 3 signal transduction in ocular Tenon’s capsule fibroblasts (OTFs). METHODS: Primary ocular Tenon’s capsule fibroblasts had been cultured in vitro. The effect of Y27632 on proliferation of OTF stimulated by lysophosphatidic acid (LPA) was evaluated by MTT colorimetric assay so as to sift out the proper concentrations range of Y-27632 for the next experiment. Real time-polymerase chain reactor (RT-PCR) was to analyze the changes of Smad2 and Smad3 genes of cells affected by Y-27632, though unaffected by transforming growth factor-beta1 (TGF-β1). Proteins of Smad2, Smad3, phosphorylated Smad2 (Ser245/250/255), and phosphorylated Smad3 (Ser423/425/203) were respectively quantified by Western blot after OTFs were successively incubated by TGF-β1 and Y-27632. Meanwhile, α-smooth muscular actin (α-SMA) protein was also quantified after the small intervening gene fragments of human Smad2 and Smad3 were designed, synthesized, and then transfected to OTFs. RESULTS: Y-27632 significantly inhibited OTFs proliferation stimulated by LPA. Also Y-27632 significantly suppressed the expressions of Smad2 mRNA, Smad2, 3 proteins expressions, Smad3 phosphorylation at the carboxylic terminals of Ser423/425/203 which had been radically promoted by TGF-β1. SiRNA-Smad2, 3 suppressed α-SMA expressions, but less effectively than Y-27632. CONCLUSION: The inhibition of ROCK signaling may be a potential therapeutic candidate for the treatment of the filtration channel fibrosis.