Background Pigment epithelium-derived factor (PEDF)–derived 34-mer peptide (PEDF34, Asp 44 -Asn 77 ) has anti-angiogenic activity but has limitations in clinical application because of an inverted bell–shaped dose-effect relationship and a short half-life. In this study, we attempted to mitigate these problems by mixing PEDF34 with type I collagen. Methods The anti-angiogenic activity of the PEDF34/atelocollagen mixture was evaluated by HUVEC tube formation assay and in a laser-induced choroidal neovascular (CNV) mouse model. PEDF34 and/or collagen were administrated using intravitreal injections or eye drops. CNV lesion size was quantified using FITC-dextran-perfused retinal whole mounts. Western blot analysis and inhibitor assays were used to define the action mechanisms of PEDF34 and the mixture. Results Collagen broadened the effective dose range of PEDF34 in the tube formation assay by > 250 times (from 0.2 to 50 nM). In the CNV model, five intravitreal injections of PEDF34 were required for therapeutic effect, whereas the mixture had a significant therapeutic effect following a single injection. Eye drops of the mixture showed significantly stronger CNV-suppressive effects than drops of PEDF34 alone. The anti-angiogenic activity of PEDF34 might be mediated by inhibition of ERK and JNK activation by VEGF, and collagen potentiated these effects. Conclusions Collagen can serve as a carrier and reservoir of PEDF34. PEDF peptide/collagen mixture is easy to prepare than conventional methods for maintaining the therapeutic effect of PEDF peptide.