We aimed to investigate the in vitro effect of pirfenidone (PFD) on proliferation, migration and collagen contraction of human pterygium fibroblasts (HPFs). HPFs were obtained from tissue explants during pterygium surgery. After treatment with pirfenidone, the HPFs proliferation was measured by MTT, cell cycle progression measured by flow cytometry, cell migration measured by the scratch assay, and cell contractility evaluated in fibroblast-populated collagen gels. The expression of TGF-beta 1, TGF-beta 2, MMP-1 and TIM P-1 were also determined with quantitative PCR, western blot and immunofluorescence staining. Results showed pirfenidone markedly inhibited HPFs proliferation with an IC50 of approximately 0.2 mg/ml. After treatment with 0.2 mg/ml pirfenidone for 24 hours, HPFs were at G0/G1 cell cycle arrest, with significantly reduced cell migration capability and collagen contraction, decreased mRNA and protein expressions of TGF-beta 1, TGF-beta 2 and MMP-1, and no alterations of TIMP-1 expression. Thus, we have concluded that pirfenidone at 0.2 mg/ml inhibits proliferation, migration, and collagen contraction of HPFs, which is associated with decreased expression of TGF-beta and MMP-1, and pirfenidone might represent a potentially therapeutic agent to prevent the recurrence of pterygium after surgery.