Elevated expression of tissue transglutaminase (TG2) in cancer cells has been implicated in the development of drug resistance and metastatic phenotypes. However, the role and the mechanisms that regulate TG2 expression remain elusive. Here, we provide evidence that protein kinase Cdelta (PKCdelta) regulates TG2 expression, which in turn inhibits autophagy, a type II programmed cell death, in pancreatic cancer cells that are frequently insensitive to standard chemotherapeutic agents. Rottlerin, a PKCdelta-specific inhibitor, and PKCdelta small interfering RNA (siRNA) down-regulated the expression of TG2 mRNA and protein and induced growth inhibition without inducing apoptosis in pancreatic cancer cells. Inhibition of PKCdelta by rottlerin or knockdown of TG2 protein by a TG2-specific siRNA resulted in a marked increase in autophagy shown by presence of autophagic vacuoles in the cytoplasm, formation of the acidic vesicular organelles, membrane association of microtubule-associated protein 1 light chain 3 (LC3) with autophagosomes, and a marked induction of LC3-II protein, important hallmarks of autophagy, and by electron microscopy. Furthermore, inhibition of TG2 by rottlerin or by the siRNA led to accumulation of green fluorescent protein (GFP)-LC3-II in autophagosomes in pancreatic cancer cells transfected with GFP-LC3 (GFP-ATG8) expression vector. Knockdown of Beclin-1, a specific autophagy-promoting protein and the product of Becn1 (ATG6), inhibited rottlerin-induced and TG2 siRNA-induced autophagy, indicating that Beclin-1 is required for this process. These results revealed that PKCdelta plays a critical role in the expression of TG2, which in turn regulates autophagy. In conclusion, these results suggest a novel mechanism of regulation of TG2 and TG2-mediated autophagy in pancreatic cancer cells.