Updated project metadata. CASPASE-3 (CASP3) is well known for its proteolytic function that mediates multiple key cell death-initiated processes and other related cellular processes. However, the possibility that CASP3 may also possess important additional non-catalytic functions in mammalian cells has remained largely unexplored. We now report the results of CASP3 knockdown, rescue, proteomic analysis and flow cytometry experiments initially in normal and malignant human mammary cells and later shown to extent to all other human cell types tested. The results reveal a new role of the CASP3 prodomain in regulating the cell cycle progression, survival, proliferation, and protein aggregate accumulation in all cells tested. The generality of these findings suggests that the ancestral pro-survival role of CASP3 in yeast persisted throughout evolution via a conservation of its prodomain, predating its later acquisition of an inherent catalytic property and subsequently preserved cell death control functions.