Retinal fibrosis, including epiretinal membrane (ERM) and proliferative vitreoretinopathy (PVR), is an ocular disease that can lead to blindness. An efficacious therapy remains an unmet medical challenge. In this study, we intended to explore the inhibitory effects of the nanoparticle-mediated delivery of plasminogen kringle 5 (K5-NPs) on laser-induced ERM and to identify the potential anti-fibrosis targets of K5-NPs. A rat model of laser-induced ERM was used. Control-NPs or K5-NPs were intravitreally injected. ERM formation was observed in vivo. The expression of fibrosis-associated factors including TGF-β, CTGF, and α-SMA was detected by qRT-PCR, Western blot, or immunohistochemistry. Our results showed that K5-NPs effectively inhibit laser-induced ERM formation. The expression of α-SMA, the marker of myofibroblast, apparently decreased in immunostained ERMs in the K5-NPs group. The expression of CTGF, a pro-fibrotic factor, in our ERM rat model was significantly downregulated by K5-NPs. Meanwhile, the expression of TGF-β, the upstream regulator of CTGF, was found to be suppressed by K5-NPs as well. The anti-fibrosis effect of K5-NPs might be achieved by interfering with TGF-β expression, thus abrogating the TGF-β-induced upregulation of CTGF and α-SMA. Our study has an important clinical relevance, demonstrating that K5-NPs might offer an additional efficacious clinical option for treating fibrosis-related diseases.