AbstractIn primary cultures of human neurons, 17β‐estradiol (17β‐E2) prevents caspase‐6‐mediated cell death and induces a caspase inhibitory factor (CIF) inhibiting active caspase‐6 (Csp‐6) in vitro. Here, we show that treatment of neurons with 17β‐E2 results in a proteasomal‐dependent but ubiquitin‐independent degradation of endogenous and exogenous active Csp‐6 in live neurons and in cell free assays, respectively. We further show that the proteasomal‐dependent degradation of Csp‐6 is not required for its inhibition. Using several protease inhibitors, we find that leupeptin, E‐64, and ALLN prevent inhibition of recombinant active Csp‐6 (R‐Csp‐6) in 17β‐E2‐treated neuronal protein extracts. Because all three protease inhibitors have the ability to inhibit cysteine proteases, we believe that a cysteinyl protease activity may be required for 17β‐E2‐mediated inhibition of active Csp‐6. However, we exclude caspases, calpains, and cathepsins as potential cysteinyl proteases involved in the 17β‐E2‐mediated Csp‐6 inhibition. The results suggest that a proteolytic activity inhibited by leupeptin, E‐64, and ALLN is needed to inhibit Csp‐6 and that the inhibited Csp‐6 is subsequently degraded by the proteasome. The mechanism of 17β‐E2‐mediated inhibition of Csp‐6 is different from the ubiquitin‐dependent proteasomal degradation of Csp‐3 and Csp‐7 by XIAP and cIAP2 but consistent with the mechanism of Baculovirus p35 inhibition of caspases.