Abstract Proteins of the BCL-2 family are important regulators of apoptosis. The BCL-2 family includes three main subgroups: the anti-apoptotic group, such as BCL-2, BCL-XL, BCL-W, and MCL-1; multi-domain pro-apoptotic BAX, BAK; and pro-apoptotic “BH3-only” BIK, PUMA, NOXA, BID, BAD, and SPIKE. SPIKE, a rare pro-apoptotic protein, is highly conserved throughout the evolution, including Caenorhabditis elegans, whose expression is downregulated in certain tumors, including kidney, lung, and breast. In the literature, SPIKE was proposed to interact with BAP31 and prevent BCL-XL from binding to BAP31. Here, we utilized the Position Weight Matrix method to identify SPIKE to be a BH3-only pro-apoptotic protein mainly localized in the cytosol of all cancer cell lines tested. Overexpression of SPIKE weakly induced apoptosis in comparison to the known BH3-only pro-apoptotic protein BIK. SPIKE promoted mitochondrial cytochrome c release, the activation of caspase 3, and the caspase cleavage of caspase’s downstream substrates BAP31 and p130CAS. Although the informatics analysis of SPIKE implicates this protein as a member of the BH3-only BCL-2 subfamily, its role in apoptosis remains to be elucidated.