Tissue transglutaminase (TG2), an enzyme involved in protein cross-linking and overexpressed in ovarian tumors, has antiapoptotic effects in cancer cells and may play a role in response to chemotherapy. In this study, we investigated the role of TG2 in the sensitivity of ovarian cancer cells to cisplatin. By using stable knockdown and overexpression strategies, we demonstrate that the level of expression of TG2 regulates apoptosis induced by cisplatin in SKOV3 and OV-90 ovarian cancer cells. Interestingly, not only TG2 knockdown but also a TG2 enzymatic inhibitor (KCC009) sensitized SKOV3 cells to cisplatin. To understand the mechanism by which TG2 exerts its antiapoptotic role, we examined the effects of protein kinase B (Akt) and nuclear factor-kappa B (NF-kappaB), two survival pathways commonly involved in development of drug resistance. Overexpression of the constitutively active p65 subunit of NF-kappaB, but not constitutively active Akt, rescued cells with diminished TG2 expression from cisplatin-induced apoptosis. This implicates activation of NF-kappaB as the main cisplatin resistance mechanism downstream of TG2. Indeed, NF-kappaB activity is decreased and the level of the inhibitory subunit I kappaB alpha is increased in ovarian cancer cells engineered to express diminished levels of TG2 or treated with the enzymatic inhibitor, KCC009. Our data show that TG2 prevents apoptosis induced by cisplatin by activating the NF-kappaB survival pathway in ovarian cancer cells.