Choroidal neovascularization (CNV) is a key pathological feature of several leading causes of vision loss including neovascular age-related macular degeneration. Here, we show that a calreticulin anti-angiogenic domain (CAD)-like peptide 27, CAD27, inhibited in vitro angiogenic activities, including tube formation, migration of endothelial cells, and vascular sprouting from rat aortic ring explants. In a rat model of laser-induced CNV, we demonstrate that intravitreal injection of CAD27 significantly attenuated the formation of CNV lesions as measured via fundus fluorescein angiography and choroid flat-mounts (19.5% and 22.4% reductions at 10 μg and 20 μg of CAD27 injected, respectively). Similarly, the reduction of CNV lesions was observed in rats that had received topical applications of CAD27 (choroid flat-mounts: 17.9% and 32.5% reductions at 10 μg/mL and 20 μg/mL of CAD27 instilled, respectively). Retinal function was unaffected, as measured using electroretinography in both groups receiving interareal injection or topical applications of CAD27 for at least fourteen days. These findings show that CAD27 can be used as a potential therapeutic alternative for targeting CNV in diseases such as neovascular age-related macular degeneration.