White adipose tissue (WAT) is the primary site in the body for storing triglycerides. WAT distribution and function vary based on sex, and accordingly, disorders in which WAT plays a role, including obesity, type two diabetes, and metabolic syndrome, have sex differences in risk factors and etiologies. Prohibitin 1 (PHB1) is a protein involved in adipocyte-intrinsic activities sensitive to sex hormone signaling. PHB1 has a bidirectional relationship with estrogens and androgens, partly mediated by tyrosine phosphorylation. Another post-translational modification site in PHB1 is Cys69. It is the only cysteine residue in the amino acid sequence of the Phb1 gene, and it is essential in targeting PHB1 to plasma membranes in adipocytes. To investigate the role of Cys69 at a systemic level, we developed a knock-in mouse model replacing Cys69 with an alanine in Phb1. These are known as the Phb1-KiC69A (or Phb1-Ki) mice. Based on what is known about PHB1, I hypothesized that these mice would display sex differences in their WAT phenotype. This study's objectives were to define changes in the WAT phenotype in the male and female Phb1-Ki mice compared to wild-type mice, and to determine whether any differences displayed sex-specificity. Visceral (VAT) and subcutaneous (SAT) adipose tissue was collected from wild-type and Phb1-Ki male and female mice. They were compared based on histology, gene and protein expression, and PHB1 subcellular localization. Primary cells from VAT and SAT were also collected and differentiated into adipocytes, and lipolysis and lipid uptake were measured. The serum concentrations of three adipokines involved in metabolic regulation were analyzed. The results showed that Phb1-Ki mice have sex- and depot-specific changes compared to wild-type mice in lipid droplet size, tissue mass, PHB1 subcellular localization, and adipocyte protein expression levels. Resistin levels are also decreased in both sexes of Phb1-Ki mice compared to wild-type. Adipocytes from VAT and SAT show no changes in lipogenesis or lipolysis in primary cultures between sex and genotype. Overall, these results show that the Phb1 Cys69 residue affects the WAT phenotype in a sex-specific manner in mice. However, this does not interrupt triglyceride synthesis or release in adipocytes in vitro.