Here, we show that recombinant bovine PDE5A1 is proteolysed by recombinant caspase‐3 in in vitro and transfected Cos‐7 cells. In addition, the treatment of PDE5A1‐transfected Cos‐7 and PC12 cells with staurosporine, an apoptotic agent that activates endogenous caspase‐3, also induced proteolysis and inactivation of PDE5A1. These findings suggest that there is specificity in the interaction between caspase‐3 and PDE5A1 that requires application of an apoptotic stimulus. The potential proteolysis of the [778]DQGD[781] site in PDE5A1 by caspase‐3 might affect cGMP's hydrolyzing activity as this is within the boundary of the active site. We therefore created a truncated D781 mutant corresponding exactly to the potential cleavage product. This mutant was expressed equally well compared with the wild‐type enzyme in transfected Cos‐7 cells and was inactive. Inactivity of the truncated mutant was not due to potential misfolding of the enzyme as it eluted from gel filtration chromatography in the same fraction as the wild‐type enzyme. Homology model comparison with the catalytic domain of PDE4B2 was used to probe a functional role for the region in PDE5A1 that might be cleaved by caspase‐3. From this, we can predict that a caspase‐3‐mediated cleavage of the [778]DQGD[781] motif would result in removal of the C‐terminal tail containing Q807 and F810, which are potentially important amino acids required for substrate binding.