Caspases are synthesised as zymogens which are cleaved and subsequently activated following specific signals. Proper regulation of caspase activation and activity is essential for their function in cell death and/or inflammation. Caspase-2 is the most evolutionarily conserved member of the caspase family and has been shown to have both apoptotic and non-apoptotic functions. Recent studies have demonstrated that activation of caspase-2 and its enzymatic activity are required in caspase-2-mediated tumour suppression. However, the molecular mechanisms that regulate caspase-2 activation and its activity remain to be fully explored. Previous studies suggest that caspase-2 activation can be modulated by phosphorylation in different experimental settings. To better understand the regulation of caspase-2 activation and activity, using an MS approach we identified six new phosphorylation sites in caspase-2. Interestingly, one of these (tentatively named S8) is highly conserved and mutagenesis studies indicated that phosphorylation at this site blocks the caspase-2 catalytic activity and its function in limiting polyploidy following mitotic insults, without affecting caspase-2 dimerisation. Molecular modelling suggests that phosphorylation at S8 may affect substrate binding by caspase-2. We propose that phosphorylation at S8 of caspase-2 is a key mechanism in regulating its catalytic activity and function.