Prohibitin-1 (PHB1) is a highly conserved protein that plays a crucial role in regulating cell proliferation, apoptosis, mitochondrial stability, and cell signaling processes. PHB1 undergoes various post-translational modifications (PTMs), enabling it to execute cell compartment-specific and pleiotropic functions. One such PTM site is cysteine 69 (Cys69) residue, which is conserved among vertebrates. Our laboratory developed a knock-in mouse model (Phb1-Ki), substituting Cys69 with an alanine residue, to explore the role of Cys69 in PHB1’s pleiotropic functions, including immune function. The Phb1-Ki mice displayed changes in spleen and thymus weights, and their histological analysis showed morphological differences when compared with age and sex matched wild-type control mice. The flow cytometry experiments revealed a decrease in CD4+ and CD8+ cell populations in the spleen of the male and in the thymus of female Phb1-Ki mice compared with respect to wild-type mice. To further understand PHB1's role in T cells, CD4+ cells isolated from the spleen were differentiated to Th1 and Th2 cells. Subsequent functional analysis showed a significant change in their marker cytokine production. Real-time PCR analysis revealed decreased mRNA levels of Phb1 and Phb2 in both (male and female) Phb1-Ki mice compared with wild-type mice. In summary, findings from Phb1-Ki mice suggest a role of Cys69-linked function in mediating PHB1’s sexually dimorphic immune function in lymphoid tissues, which may in part be mediated through its homologue and heterodimeric partner protein PHB2 because of their multifaceted relationship with sex steroids.