Transglutaminase 2 (TG2) is a multifunctional enzyme that has been implicated in the pathogenesis of neurodegenerative diseases, ischemia, and stroke. The mechanism by which TG2 modulates disease progression have not been elucidated. In this study we investigate the role of TG2 in the cellular response to ischemia and hypoxia. TG2 is up-regulated in neurons exposed to oxygen and glucose deprivation (OGD), and increased TG2 expression protects neurons against OGD-induced cell death independent of its transamidating activity. We identified hypoxia inducible factor 1 (HIF1) as a TG2 binding partner. HIF1 and HIF1 together form the heterodimeric transcription factor hypoxia inducible factor 1 (HIF1). TG2 and the transaminase-inactive mutant C277S-TG2 inhibited a HIF-dependent transcription reporter assay under hypoxic conditions without affecting nuclear protein levels for HIF1 or HIF1, their ability to form the HIF1 heterodimeric transcription factor, or HIF1 binding to its DNA response element. Interestingly, TG2 attenuates the up-regulation of the HIF-dependent proapoptotic gene Bnip3 in response to OGD but had no effect on the expression of VEGF, which has been linked to prosurvival processes. This study demonstrates for the first time that TG2 protects against OGD, interacts with HIF1, and attenuates the HIF1 hypoxic response pathway. These results indicate that TG2 may play an important role in protecting against the delayed neuronal cell death in ischemia and stroke.—Filiano, A. J., Bailey, C. D. C., Tucholski, J., Gundemir, S., Johnson, G. V. W. Transglutaminase 2 protects against ischemic insult, interacts with HIF1, and attenuates HIF1 signaling.