Neuroglobin (Ngb) is a recently discovered heme protein in the vertebrate brain that can bind to oxygen molecules. Mammalian Ngb plays a crucial role in neuroprotection under conditions of oxidative stress. To investigate other potential functions of Ngb, we investigated the mouse retinal Ngb system following optic nerve injury. In the retina of control mice, Ngb immunoreactivity was limited to the retinal ganglion cell (RGC) layer, and this immunoreactivity rapidly decreased to less than 50% of the control level 5 days after optic nerve injury. On the basis of this decrease, we designed in vivo experiments with enhanced expression of Ngb using adult mouse retina. The enhanced expression of Ngb was achieved by injecting chimeric human Ngb protein, which included the cell membrane-penetrating module of fish Ngb. One-day pretreatment with chimeric Ngb increased immunoreactivity levels of Ngb two-fold in mouse RGCs and increased the number of surviving RGCs three-fold by 14 days after optic nerve injury compared with vehicle controls. Furthermore, in the mouse retinas showing enhanced Ngb expression, several regenerating central optic axons exhibited outgrowth and were found to pass through the nerve crush site 14 days after nerve injury. No such regenerating optic axons were observed in the control mouse optic nerve during the same time frame. The data obtained from in vivo experiments strongly indicate that mammalian Ngb has neuroprotective and neuroregenerative properties.