Caffeine is a globally consumed psychostimulant but can be fatal to cells at overdose exposures. Although caspase-dependent apoptosis plays a role in caffeine-induced cell death, the responsible intracellular signalling cascade remains incompletely understood. The cellular slime mould, Dictyostelium discoideum, does not possess caspase-dependent apoptotic machinery. Here, we observed that ablation of D. discoideumplaA, which encodes a phospholipase A2 (PLA₂) homolog, leads to a decreased rate of cell death under high caffeine concentrations and to enhanced cell death with the addition of arachidonic acid. Moreover, the inhibition of PLA₂ activity lead to a recovery of the survival rate in caspase-inhibited Hela cervical carcinoma cells under high caffeine concentrations, indicating that caffeine-induced cell death is enhanced via PLA₂-dependent signalling. Our results indicate that arachidonic acid may be a general second messenger that negatively regulates caffeine tolerance via a caspase-independent cell death cascade, which leads to multiple effects in eukaryotic cells.