Autophagy is mostly a nonselective bulk degradation system within cells. Recent reports indicate that autophagy can act both as a protector and killer of the cell depending on the stage of the disease or the surrounding cellular environment (for review see Cuervo, A.M. 2004. Trends Cell Biol. 14:70–77). We found that cytoplasmic vacuoles induced in pancreatic acinar cells by experimental pancreatitis were autophagic in origin, as demonstrated by microtubule-associated protein 1 light chain 3 expression and electron microscopy experiments. To analyze the role of macroautophagy in acute pancreatitis, we produced conditional knockout mice lacking the autophagy-related 5 gene in acinar cells. Acute pancreatitis was not observed, except for very mild edema in a restricted area, in conditional knockout mice. Unexpectedly, trypsinogen activation was greatly reduced in the absence of autophagy. These results suggest that autophagy exerts devastating effects in pancreatic acinar cells by activation of trypsinogen to trypsin in the early stage of acute pancreatitis through delivering trypsinogen to the lysosome.