A cytotoxic enterotoxin (Act) of Aeromonas hydrophila possesses several biological activities, induces an inflammatory response in the host, and causes apoptosis of murine macrophages. In this study, we utilized five target cell types (a murine macrophage cell line (RAW 264.7), bone marrow-derived transformed macrophages, murine peritoneal macrophages, and two human intestinal epithelial cell lines (T84 and HT-29)) to investigate the effect of Act on mitogen-activated protein kinase (MAPK) pathways and mechanisms leading to apoptosis. As demonstrated by immunoprecipitation/kinase assays or Western blot analysis, Act activated stress-associated p38, c-Jun NH(2)-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2) in these cells. Act also induced phosphorylation of upstream MAPK factors (MAPK kinase 3/6 (MKK3/6), MKK4, and MAP/ERK kinase 1 (MEK1)) and downstream effectors (MAPK-activated protein kinase-2, activating transcription factor-2, and c-Jun). Act evoked cell membrane blebbing, caspase 3-cleavage, and activation of caspases 8 and 9 in these cells. In macrophages that do not express functional tumor necrosis factor receptors, apoptosis and caspase activities were significantly decreased. Immunoblotting of host whole cell lysates revealed Act-induced up-regulation of apoptosis-related proteins, including the mitochondrial proteins cytochrome c and apoptosis-inducing factor. However, mitochondrial membrane depolarization was not detected in response to Act. Taken together, the data demonstrated for the first time Act-induced activation of MAPK signaling and classical caspase-associated apoptosis in macrophages and intestinal epithelial cells. Given the importance of MAPK pathways and apoptosis in inflammation-associated diseases, this study provided new insights into the mechanism of action of Act on host cells.