The multifunctional caspase-2 protein is involved in apoptosis, NF-κB regulation, and tumor suppression in mice. However, the mechanisms of caspase-2 responsible for tumor suppression remain unclear. Here we identified two sites of caspase-2, the catalytic Cys-320 site and the Ser-139 site, to be important for suppression of cellular transformation and tumorigenesis. Using SV40- and K-Ras-transformed caspase-2 KO mouse embryonic fibroblast cells reconstituted with expression of wild-type, catalytic dead (C320A), or Ser-139 (S139A) mutant caspase-2, we demonstrated that similar to caspase-2 deficiency, when Cys-320 and Ser-139 were mutated, caspase-2 lost its ability to inhibit cellular transformation and tumorigenesis. These mutant cells exhibited enhanced cell proliferation, elevated clonogenic activity, accelerated anchorage-independent growth, and transformation and were highly tumorigenic, rapidly producing large tumors in athymic nude mice. Investigation into the underlying mechanism showed that these two residues are needed for caspase-2 to suppress NF-κB activity, promote apoptosis, and sustain the G(2)/M checkpoint following DNA damage induction. In addition, tumors in nude mice derived from the two mutant cell lines had higher constitutive NF-κB activity and elevated expression of NF-κB targets of antiapoptotic proteins Bcl-xL, XIAP, and cIAP2. A reduction in caspase-2 mRNA was associated with multiple types of cancers in patients. Together, these observations suggest the combined functions of caspase-2 in suppressing NF-κB activation, promoting apoptosis, and sustaining G(2)/M checkpoint contribute to caspase-2 tumor-suppressing function and that caspase-2 may also impact tumor suppression in humans. These findings provide insight into tumor suppression at the cross-roads of apoptosis, cell cycle checkpoint, and NF-κB pathways.