Listeriolysin O (LLO) is a cholesterol-dependent cytolysin that mediates escape of from phagosomes and enables the bacteria to grow within the host. LLO is a versatile tool allowing to trigger several cellular responses. In this study, rapid phosphorylation of ERK1/2 on Caco-2 cells caused by infection was demonstrated to be highly dependent on LLO activity. The effect could be strongly induced by adding purified recombinant LLO alone and could be inhibited by exogenous cholesterol. Lack of the PEST sequence, known to tightly control cytotoxicity of LLO, did not affect ERK1/2 activation. However, the recombinant non-cytolytic LLO, with mutations in the cholesterol-binding motif, was unable to trigger this response. Recombinant LLO, which lacks most of the cytolytic activity, also failed to activate ERK1/2 phosphorylation, and this effect could be rescued when the protein concentration reached a cytolytic level. Infection with an LLO-deficient mutant (Δ) or the mutant complementing LLO abrogated the capacity of the bacteria to activate ERK1/2. However, infection with the Δ mutant complementing LLO, which retained partial pore-forming ability and could grow intracellularly, was capable of triggering ERK1/2 phosphorylation. Collectively, these data suggest that ERK1/2 activation by depends on the permeabilization activity of LLO and more importantly correlates with the cholesterol-binding motif of LLO.