Transglutaminase 2 (TGase 2) promotes nuclear factor-κB (NF-κB) activity through depletion of the inhibitory subunit of NF-κB (I-κBα) via protein cross-linking, leading to resolution of inflammation. Increased expression of TGase 2 contributes to inflammatory disease pathogenesis via constitutive NF-κB activation. Conversely, TGase 2 inhibition often reverses inflammation in animal models. The role of TGase 2 in apoptosis remains less clear, as both pro- and anti-apoptotic functions of TGase 2 have been demonstrated under different experimental conditions. Apoptosis is intact in a TGase 2 knock out mouse (TGase2(-/-)), which is phenotypically normal. However, upon exposure to tumor necrosis factor (TNF)-α-induced apoptotic stress, mouse embryonic fibroblasts (MEFs) from TGase2(-/-) mice were more sensitive to cell death than MEFs from wild-type (TGase 2(+/+)) mice. In the current study, to explore the role of TGase 2 in apoptosis, TGase 2-binding proteins were identified by LC/MS. TGase 2 was found to associate with cathepsin D (CTSD). Binding of TGase 2 to CTSD resulted in the depletion of CTSD via cross-linking in vitro as well as in MEFs, leading to decreased levels of apoptosis. Furthermore, cytoplasmic CTSD levels were higher in MEFs from TGase 2(-/-) mice than in those from TGase 2(+/+) mice, as were caspase 3 activation and poly (ADP-ribose) polymerase (PARP) processes. These results suggest that TGase 2, while not previously implicated as a major regulatory factor in apoptosis, may regulate the balance between cell survival and cell death through the modulation of CTSD levels.