Apoptosis plays a key role in the maintenance of a constant cell number and a low incidence of cancer in the mucosa of intestine. Although the small GTPase Rac1 has been established as an important regulator of migration of intestinal epithelial cells, whether Rac1 is also involved in apoptosis is unclear. The current studies test the hypothesis that Rac1 mediates TNF--induced apoptosis in IEC-6 cells. Rac1 is activated during TNF--induced apoptosis as judged by the level of GTP-Rac1, the level of microsomal membrane-associated Rac1, and lamellipodia formation. Although expression of a constitutive active Rac1 does not increase apoptosis in the basal condition, inhibition of Rac1 either by NSC23766 (Rac1 inhibitor) or a dominant negative Rac1 protects cells from TNF--induced apoptosis by inhibiting caspase-3, -8 and -9. Inhibition of Rac1 prior to apoptotic stimuli significantly prevents TNF--induced activation of JNK1/2. Inhibition of Rac1 does not modulate TNF--induced ERK1/2 and Akt activation. Inhibition of ERK1/2 and Akt activity increased TNF--induced apoptosis. However, inhibition of Rac1 significantly decreased apoptosis in the presence of ERK1/2 and Akt inhibitors, similar to the effect observed with NSC23766 alone in response to TNF-. Thus Rac1 inhibition protects cells independently of ERK1/2 and Akt activation during apoptosis. Although, p38 MAP kinase is activated in response to TNF-, inhibition of p38 MAP kinase does not decrease apoptosis. Rac1 inhibition does not alter p38 MAP kinase activity. Thus, these results indicate that Rac1 mediates apoptosis via JNK and plays a key role in pro-apoptotic pathways in intestinal epithelial cells.