Retinal ischemia reperfusion injury (IRI) is a leading cause of visual impairment or blindness, and an effective way to prevent the visual loss needs to be developed. Although decades of clinical application of Huoxue-Tongluo-Lishui-Decoction (HTLD) has demonstrated its reliable clinical efficacy against retinal IRI, no convincing randomized controlled trials were conducted in humans or animals, and the associated mechanism still needs to be explored. To confirm the protective effect of HTLD against retinal IRI and to explore its underlying mechanisms, a standard retinal IRI animal model, randomized controlled trials, objective evaluation and examination methods were adopted in this study. Flash visual evoked potentials (F-VEP) was performed 8 weeks post-reperfusion. The results showed that the medium dose of HTLD had better treatment effects than low dose of HTLD. High dose of HTLD did not further improve visual function relative to medium dose of HTLD, but had poor performance in the latency of P2 wave. The angio-optical coherence tomography (angio-OCT) examination showed that retinal nerve fiber layer (RNFL) became edematous in the early stage, then the edema subsided, and RNFL became thinning in the late stage. HTLD reduced the swelling of RNFL in the early stage and prevented the thinning of RNFL in the late stage. Similar to F-VEP, medium dose of HTLD has the best neural-protective effects against retinal IRI. In mechanisms, HTLD treatment not only enhanced autophagy at 6 h after reperfusion, but extended the enhancing effect until at least 24 h. HTLD treatment significantly reduced the cleaved Caspase-3, cleaved PARP and Caspase-3 activity at 48 h after reperfusion. HTLD inhibited neuro-toxic cytokines expression in retinal IRI by modulating Akt/NF-kB signaling. HTLD treatment enhanced the expressions of L-glutamate/L-aspartate transporter (GLAST) and glutamine synthetase (GS), and lower the concentration of free glutamate in retina after reperfusion. The phosphorylation of iNOS increased significantly in retinal IRI at 6 h, and HTLD treatment suppressed the phosphorylation of Inducible nitric oxide synthetase (iNOS). In conclusion, HTLD is visual-protective against retinal IRI, and the regulation of autophagy, apoptosis and neuro-toxic mediators may be the underlying mechanisms. These findings may provide new ideas for the clinical treatment of retinal IRI related diseases.