Programmed cell death (PCD) is a physiological cell death under the control of genetic mechanisms. In animals, apoptosis has been thoroughly elucidated at the molecular level. Cysteine aspartic acid specific proteases termed 'caspases' are central to the apoptotic initiation and execution in animals. However, although caspase-like enzymatic activities are required for plant PCD execution, there is no caspase homologue with caspase activity known in plants. Thus the search for caspase functional analogues is an important approach to improve the understanding of the regulatory mechanisms underlying plant PCD. The protease cathepsin B (AtCathB) was identified in fractions of partially purified caspase-3-like activities extracted from Arabidopsis seedlings after UV-C induction. In addition, an elevated cathepsin B-like enzymatic activity coinciding with the elevated caspase-3-like enzymatic activity was detected in Arabidopsis in response to oxidative stresses. Consequently, AtCathB double knockout and triple knockout transgenic lines were generated using T-DNA insertion as well as artificial microRNAs mediated gene silencing. AtCathB loss-of-function lines showed an abolished PCD triggered by oxidative stresses and a significant reduced cathepsin B-like activity and caspase-3-like activity.Three AtCathB genes are present in the Arabidopsis genome and recombinant AtCathB-2 and AtCathB-3 were produced using E.coli and insect cell expression systems. Results from proteolytic activity assay and kinetic measurement confirmed the enzymatic similarity between human caspase-3 and Arabidopsis cathepsin B. Preliminary experiments aimed at identifying cathepsin B in vivo substrates have identified the enzyme RuBisCO as a strong candidate. At the subcellular level, AtCathB-2 was found to be localised in the vacuole and AtCathB-3 mainly in the apoplast. In summary, Arabidopsis cathepsin B has caspase-3-like activity and regulates oxidative stress-induced PCD. The possible conservation through evolution from plants to animals of an ancestral cathepsins-mediated apoptosis/PCD regulatory pathway is discussed.