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Caspase-3 is responsible for the cleavage of several proteins including the nuclear enzyme poly(ADP-ribose) polymerase (PARP). Designed on the cleavage site of PARP, Ac-Asp-Glu-Val-Asp-H has been reported as a highly specific inhibitor. To overcome the susceptibility to proteolysis, the intrinsic instability, and the scarce membrane permeability of tetra-peptidyl aldehydes, di- and tri-peptidyl caspase-3 inhibitors have been synthesized. Here, the synthesis and the inhibition properties of peptidyl aldehydes Z- t Leu-Asp-H, Z- t Leu-Val-Asp-H, and Z-Val- t Leu-Asp-H are reported. Z- t Leu-Asp-H, Z- t Leu-Val-Asp-H, and Z-Val- t Leu-Asp-H inhibit competitively human caspase-3 activity in vitro with K i 0 = 3.6 nM, 18.2 nM, and 109 nM, respectively (pH 7.4 and 25 °C). Moreover, Z- t Leu-Asp-H impairs apoptosis in human DLD-1 colon adenocarcinoma cells without affecting caspase-8. Therefore, Ac-Asp-Glu-Val-Asp-H can be truncated to Z- t Leu-Asp-H retaining nanomolar inhibitory activity in vitro and displaying action in whole cells, these properties reflect the unprecedented introduction of the bulky and lipophilic t Leu residue at the P 2 position.