Entamoeba histolytica virulence has been attributed to several amoebic molecules such as adhesins, amoebapores and cysteine proteinases, but supporting evidence is either partial or indirect. In this work we compared several in vitro and in vivo features of both virulent E. histolytica (vEh) and non-virulent E. histolytica (nvEh) axenic HM-1 IMSS strains, such as complement resistance, proteinase activity, haemolytic, phagocytic and cytotoxic capacities, survival in mice caecum, and susceptibility to O(2). The only difference observed was a higher in vitro susceptibility of nvEh to O(2). The molecular mechanism of that difference was analyzed in both groups of amoebae after high O(2) exposure. vEh O(2) resistance correlated with: (i) higher O(2) reduction (O(2)(-) and H(2)O(2) production); (ii) increased H(2)O(2) resistance and thiol peroxidase activity, and (iii) reversible pyruvate: ferredoxin oxidoreductase (PFOR) inhibition. Despite the high level of carbonylated proteins in nvEh after O(2) exposure, membrane oxidation by reactive oxygen species was not observed. These results suggest that the virulent phenotype of E. histolytica is related to the greater ability to reduce O(2) and H(2)O(2) as well as PFOR reactivation, whereas nvEh undergoes irreversible PFOR inhibition resulting in metabolic failure and amoebic death.