Linking gender-specific differences to the molecular etiology of obesity has been largely based on genomic and transcriptomic evidence lacking endophenotypic insight and is not applicable to the extracellular fluid compartments, or the milieu intérieur, of the human body. To address this need, this study profiled the whole serum proteomes of age-matched nondiabetic overweight and obese females (n = 28) and males (n = 31) using a multiplex design with pooled biological and technical replicates. To bypass basic limitations of immunodepletion-based strategies, subproteome enrichment by size-exclusion chromatography (SuPrE-SEC) followed by iTRAQ 2D-LC-nESI-FTMS analysis was used. The study resulted in the reproducible analysis of 2472 proteins (peptide FDR < 5%, q < 0.05). A total of 248 proteins exhibited significant modulation between men and women (p < 0.05) that mapped to pathways associated with β-estradiol, lipid and prostanoid metabolism, vitamin D function, immunity/inflammation, and the complement and coagulation cascades. This novel endophenotypic signature of gender-specific differences in whole serum confirmed and expanded the results of previous physiologic and pharmacologic studies exploring sexual dimorphism at the genomic and transcriptomic level in tissues and cells. Conclusively, the multifactorial and pleiotropic nature of human obesity exhibits sexual dimorphism in the circulating proteome of importance to clinical study design.