Caspase-6 (Casp6) is a short pro-domain caspase that is activated early in Alzheimer disease, yet, little is known on the mechanism of activation of this caspase. In this study, critical proteolytic processing events required for Casp6 activation in vitro and in vivo were evaluated by site directed mutagenesis of the D23 pro-domain, and D179 and D193 linker processing sites. We found that (1) Casp6 was self-processed and activated in vitro and in vivo, (2) uncleavable Casp6 possessed low activity in vitro but not in vivo, (3) the pro-domain of Casp6 entirely prevented self-processing and activation in vivo but not in vitro, (4) removal of the pro-domain promoted Casp6 activation, (5) cleavage at either D179 or D193 was sufficient to generate activity in vitro and in vivo, and (6) Casp6 activity did not induce cell death in HEK293T cells. We conclude that the Casp6 is activated through proteolytic cleavage, as are the effector Caspase-3 and -7. However, unlike other effector caspases, Casp6 can be entirely self-activated and its activation does not necessarily induce cell death.