Ceramides are sphingolipids that have been shown to regulate several cellular processes including differentiation, growth suppression, cell senescence, and apoptosis. The cardiomyoblast cells (H9C2) were incubated with the synthetic short chain and cell permeable C2 and C6 ceramides, or the long chain C16 ceramide for different time durations. Cell viability, mitochondrial function and some markers of apoptosis were analysed using multiple complementary techniques. Our results revealed a significant reduction of H9C2 cells viability following incubation with any of the three ceramides for 24 hrs. All the three ceramides showed reductions in the mitochondrial membrane potential, and an increased cytosolic cytochrome c as a sign of mitochondrial induced apoptosis. Moreover, treatment of the cells with C6 ceramide did result in a significant reduction of mitochondrial complex I, complex I+III, complex III and complex IV activities. After one hour of ceramides treatment, the cells showed a significant induction of the proapoptotic Bcl-2 family member Bax, this was decreased after 24 hrs. Surprisingly, the cells also showed an increased induction of the antiapoptotic Bcl-xL with an insignificant splicing towards the proapoptotic Bcl-xS. Furthermore, there was increased caspase-9 activity and decreased uncleaved procaspase-3 and 9 following treatment with ceramides, probably as a sign of increased consumption of procaspase-3 and 9. In conclusion, incubation of H9C2 cells with C2, C6 or C16 ceramides resulted in a programmed cell death with atypical features of apoptosis.