IEX-1 (immediate early response gene X-1) is a stress-inducible gene. Its overexpression can either suppress or enhance apoptosis dependent of the nature of a stress, yet the polypeptide does not possess any of the functional domains that are homologous to those present in well-studied effectors or inhibitors of apoptosis. Sequence-targeting mutagenesis study reveals a transmembrane-like integrated region of the protein to be critical for both pro-apoptotic and anti-apoptotic functions. Substitution of the key hydrophobic residues with hydrophilic ones within this region severely impairs its capacity to positively and negatively regulate apoptosis. Mutations at post-translational modification sites including N-linked glycosylation and phosphorylation or truncation of IEX-1’s C-terminus also abrogated its potential to promote cell survival. However, distinguished from the transmembrane-like domain, these mutants preserved pro-apoptosis activity fully. On the contrary, mutation of nuclear localization sequence, in spite of its importance in apoptosis, did not impede IEX-1-mediated cell survival. Strikingly, all the mutants that lose their anti-apoptotic ability are unable to prevent acute increases in production of intracellular reactive oxygen species (ROS) at an initial onset of apoptosis. In contrast, mutants that can sustain anti-death function also control acute ROS production as sufficiently as WT IEX-1. These findings suggest a critical role of IEX-1 in regulation of intracellular ROS homeostasis, providing new insight into the mechanism underlying IEX-1-mediated cell survival.