This study evaluated the role of crystallins in retinal degeneration induced by chemical hypoxia. Wild-type, αA-crystallin (−/−), and αB-crystallin (−/−) mice received intravitreal injection of 12nmol (low dose), 33nmol (intermediate dose) or 60nmol (high dose) cobalt chloride (CoCl2). Hematoxylin and eosin and TdT-mediated dUTP nick-end labeling (TUNEL) stains were performed after 24h, 96h, and 1week post-injection, while immunofluorescent stains for αA- and αB-crystallin were performed 1week post-injection. The in vitro effects of CoCl2 on αB-crystallin expression in ARPE-19 cells were determined by real time RT–PCR, Western blot, and confocal microscopy and studies evaluating subcellular distribution of αB-crystallin in the mitochondria and cytosol were also performed. Histologic studies revealed progressive retinal degeneration with CoCl2 injection in wild-type mice. Retinas of CoCl2 injected mice showed transient increased expression of HIF-1α which was maximal 24h after injection. Intermediate-dose CoCl2 injection was associated with increased retinal immunofluorescence for both αA- and αB-crystallin; however, after high-dose injection, increased retinal degeneration was associated with decreased levels of crystallin expression. Injection of CoCl2 at either intermediate or high dose in αA-crystallin (−/−) and αB-crystallin (−/−) mice resulted in much more severe retinal degeneration compared to wild-type eyes. A decrease in ARPE-19 total and cytosolic αB-crystallin expression with increasing CoCl2 treatment and an increase in mitochondrial αB-crystallin were found. We conclude that lack of α-crystallins accentuates retinal degeneration in chemically induced hypoxia in vivo.