The cyclooxygenase (COX)-2 inhibitor Celecoxib may inhibit cancer cell growth independently of its capacity to block the COX-2 enzyme. The growth inhibitory effect had been attributed to its pro-apoptotic effects. However, the molecular details of Celecoxib-induced apoptosis have not been analyzed yet. To differentiate between death receptor and mitochondrial signaling pathways, induction of apoptosis upon treatment with Celecoxib was tested in Jurkat T- and BJAB B-lymphoma cell lines with defects in either pathway. Celecoxib-induced dose- and time-dependent apoptosis in Jurkat and BJAB cells involving i) activation of caspases-9, -8, and -3, ii) cleavage of poly(ADP-ribose) polymerase and inhibitor of caspase-activated DNAase, iii) breakdown of the mitochondrial membrane potential, and iv) release of cytochrome c. Lack of Fas-associated death domain protein (FADD), overexpression of a dominant negative FADD, lack of caspase-8, and treatment with caspase-8-specific inhibitors had no influence on Celecoxib-induced apoptosis. In contrast, overexpression of a dominant negative caspase-9 or pharmacological inhibition of caspase-9 strongly interfered with Celecoxib-induced cell death. Furthermore, expression of Apaf-1 was required for Celecoxib-induced apoptosis. Importantly, Bcl-2 overexpression did not abrogate caspase activation, mitochondrial alterations, and apoptosis upon Celecoxib treatment while inhibiting radiation induced apoptosis. In conclusion, Celecoxib induces apoptosis via a novel apoptosome-dependent but Bcl-2-independent mitochondrial pathway.