Hepatoprotection mediated by free radical scavenging molecules such as dimethyl sulfoxide (Me2SO) arose the question as to whether this effect involved one or several anti-apoptotic signals. Here, using primary cultures of rat hepatocytes and in vivo thioacetamide-induced liver failure, we showed that Me2SO failed to prevent any cleavage of initiator caspase-8 and -9 but constantly inhibited procaspase-3 maturation and apoptosis execution, pointing to an efficient inhibition of cleaved initiator caspase activities. Evidence was recently provided that apoptosis might require both caspase and ASK1/JNK-p38 activities. We demonstrated that this kinase pathway was strongly inhibited in the presence of Me2SO whereas overexpression of ASK1 was able to restore caspase-3 activity and apoptosis. Interestingly, we also found that GST M1/2 and GST A1/2 dropped under apoptotic conditions; furthermore transfection of GST M1, A1, or P1 to cells overexpressing ASK1, abolished caspase-3 activity and restored viability. This role of GSTs was further assessed by showing that their high expression level was tightly associated with inhibition of ASK1 activity in Me2SO-protected hepatocytes. Together, these results demonstrate that Me2SO-mediated hepatoprotection involves a dual inhibition of cleaved initiator caspase and ASK1/JNK-p38 activities. Furthermore, in highlighting the control of apoptosis by GSTs, these data provide new insights for analyzing the complex mechanisms of hepatoprotection.