In this study, the release of mitochondrial proapoptotic intermembrane space proteins induced by ex-ogenous C2-ceramide in human colon carcinoma (HT-29) cell line was investigated. HT-29 cells were treated with 12.5, 25 and 50 μmol/L C2-ceramide in vitro. Flow cytometer was used to detect the mito-chondrial membrane potential (ΔΨm). Subcellular fractions were extracted by Mitochondrial/Cytosol Fractionation Kit after C2-ceramide treatment for 24 h. SDS-PAGE was used to determine the level of cytochrome c (Cyt c), high temperature requirement A2 (HtrA2) and second mitochondrial-derived ac-tivator of caspases (Smac) released from mitochondria, the expression of X-linked inhibitor of apop-tosis protein (XIAP) and caspase-3 for 24 h. The results showed that ΔΨm began to decrease from 6 h after 25 and 50 μmol/L C2-ceramide treatment (P<0.05) and cyclosporin A (CsA) could inhibit the col-lapse of ΔΨm through regulating mitochondrial membrane permeability transition pore. There was no effect of C2-ceramide on the expression of Cyt c, HtrA2 and Smac in the total levels. 12.5, 25 and 50 μmol/L C2-ceramide could induce Cyt c, HtrA2 and Smac to release from mitochondria to cytosol and down-regulate the expression of XIAP (P<0.05). Also there was expression of cleaved caspase-3 with C2-ceramide treatment. After the treatment with caspase inhibitor, C2-ceramide still induced the release of Cyt c and HtrA2, but Smac did not. Therefore, C2-ceramide could induce apoptosis of HT-29 cells through the mitochondria pathway. The release of Cyt c, HtrA2 and Smac from mitochondria did not occur via the same mechanism, the release of Cyt c and HtrA2 was caspase-independent and the re-lease of Smac was caspase-dependent. In this study, the release of mitochondrial proapoptotic intermembrane space proteins induced by ex-ogenous C2-ceramide in human colon carcinoma (HT-29) cell line was investigated. HT-29 cells were treated with 12.5, 25 and 50 μmol / -ceramide in vitro. Flow cytometer was used to detect the mito-chondrial membrane potential (ΔΨm). Subcellular fractions were extracted by Mitochondrial / Cytosol Fractionation Kit after C2-ceramide treatment for 24 h. SDS-PAGE was used to determine the level of cytochrome c (Cyt c), high temperature requirement A2 (HtrA2) and second mitochondrial-derived ac-tivator of caspases (Smac) released from mitochondria, the expression of X-linked inhibitor of apoptin protein (XIAP) and caspase-3 for 24 h. The results showed that ΔΨm began to decrease from 6 h after 25 and 50 μmol / L C2-ceramide treatment (P <0.05) and cyclosporin A (CsA) could inhibit the col-lapse of ΔΨm through regulating mitochondrial membrane permeability transition pore. There was no effect of C2-ceramide on the expression of Cyt c, HtrA2 and Smac in the total levels. 12.5, 25 and 50 μmol / L C2-ceramide could induce Cyt c, HtrA2 and Smac to release from mitochondria to cytosol and down-regulate the expression of XIAP (P <0.05). Also there was expression of cleaved caspase-3 with C2-ceramide treatment. After the treatment with caspase inhibitor, C2-ceramide still induced the release of Cyt c and HtrA2, but Smac did not. , C2-ceramide could induce apoptosis of HT-29 cells through the mitochondria pathway. The release of Cyt c, HtrA2 and Smac from mitochondria did not occur via the same mechanism, the release of Cyt c and HtrA2 was caspase-independent and the re -lease of Smac was caspase-dependent.