We have previously demonstrated that matrix metalloprotease-3 (MMP-3) can act inside the cell to trigger apoptosis in response to various cell stresses in dopaminergic neuronal cells. However, the mechanism by which MMP-3 activity leads to caspase-3 activation in apoptotic signaling was not known. In the present study, we found that MMP-3 acts upstream of caspase-9. Overexpression of wild type MMP-3, but not mutant MMP-3, generated the enzymatically active 35kD caspase-9. The caspase-9 activation was absent in MMP-3 knockout cells, but was present when these cells were transfected with wild type MMP-3 cDNA. It was elevated in cells that were under a MMP-3-inducing ER stress condition, and this was attenuated by pharmacologic inhibition and gene knockdown of MMP-3. Incubation of recombinant catalytic domain of MMP-3 (cMMP-3) with procaspase-9 was not sufficient to cause caspase-9 activation, and an additional cytosolic factor was required. cMMP-3 was found to bind to the cytosolic protein Apaf-1, as determined by changes in surface plasmon resonance, and to cleave Apaf-1. Pharmacological inhibition, knockout, and knockdown of MMP-3 attenuated the cleavage. Taken together, the present study demonstrates that MMP-3 leads to caspase-9 activation and suggests that this occurs indirectly via a cytosolic protein, possibly involving Apaf-1.