Purpose. To investigate the effect of pirfenidone, a novel antifibrotic agent, on proliferation, migration, and collagen contraction of human Tenon’s fibroblasts (HTFs). methods. After treatment of HTFs with pirfenidone, cell proliferation was measured by MTT assay. Cell migration was investigated by scratch assay. Contractility was evaluated in fibroblast-populated collagen gels. Cell viability was determined by trypan blue exclusion assay. The expression of TGF-β1, -β2, and -β3 was estimated with RT-PCR, Western blot, and immunofluorescence analyses. results. Pirfenidone induced significant dose-dependent inhibition of HTF proliferation and migration and collagen contraction. After treatment with different concentrations of pirfenidone (0.15, 0.3, and 1 mg/mL) for 24 and 72 hours, cell viability was not different in the treatment and control groups. After 24 hours of treatment with pirfenidone, HTFs showed dose-dependent decreases in mRNA and protein levels of TGF-β1, -β2, and -β3. conclusions. These findings indicate that pirfenidone inhibits proliferation, migration, and collagen contraction of HTFs at nontoxic concentrations. A decrease in autocrine TGF-β signaling may have a role in the effects of pirfenidone.