The transcription factor nuclear factor-kappaB (NF-kappaB) plays an important role in regulating cell growth, apoptosis, and metastatic functions. Constitutive activation of NF-kappaB has been observed in various cancers; however, molecular mechanisms resulting in such activation remain elusive. Based on our previous results showing that drug-resistant and metastatic cancer cells have high levels of tissue transglutaminase (TG2) expression and that this expression can confer chemoresistance to certain types of cancer cells, we hypothesized that TG2 contributes to constitutive activation of NF-kappaB. Numerous lines of evidence showed that overexpression of TG2 is linked with constitutive activation of NF-kappaB. Tumor cells with overexpression of TG2 exhibited increased levels of constitutively active NF-kappaB. Activation of TG2 led to activation of NF-kappaB; conversely, inhibition of TG2 activity inhibited activation of NF-kappaB. Similarly, ectopic expression of TG2 caused activation of NF-kappaB, and inhibition of expression of TG2 by small interfering RNA abolished the activation of NF-kappaB. Our results further indicated that constitutive NF-kappaB reporter activity in pancreatic cancer cells is not affected by dominant-negative I kappaB alpha. Additionally, coimmunoprecipitation and confocal microscopy showed that I kappaB alpha is physically associated with TG2. Lastly, immunohistochemical analysis of pancreatic ductal carcinoma samples obtained from 61 patients further supported a strong correlation between TG2 expression and NF-kappaB activation/overexpression (P = 0.0098, Fisher's exact test). We conclude that TG2 induces constitutive activation of NF-kappaB in tumor cells via a novel pathway that is most likely independent of I kappaB alpha kinase. Therefore, TG2 may be an attractive alternate target for inhibiting constitutive NF-kappaB activation and rendering cancer cells sensitive to anticancer therapies.