Background: Research has been conducted on the dysfunction of visceral adipose tissue (VAT) in obesity, which has revealed changes in the secretion of adipokines that impact insulin resistance (IR), inflammation, endothelial dysfunction, and thrombophilia. Hypoxia is an early trigger for VAT dysfunction. In a previous study, we analyzed the proteins that are differentially expressed in the secretome of hypoxic vs normoxic 3T3-L1 murine differentiated adipocytes using nLC/MS/MS. Prothymosin-alpha (ProT-α) was identified as one of the biomolecules that is upregulated in the secretome of hypoxic adipocytes. Given ProT-α’s proposed role in immune regulation, including the orchestration of Th1 adaptive immune responses and promotion of pro-inflammatory cytokine production, and the limited information on circulating ProT-α levels in obesity, our present investigation aimed to explore its potential involvement in obesity-related inflammation and IR. Methods and results: In a case-control observational study involving 61 euglycemic obese patients and 51 lean, age-matched controls, serum samples were assayed for ProT-α levels using ELISA and a panel of inflammatory cytokines and growth factors via a multiparametric chemiluminescence microarray. Significantly elevated fasting insulin levels (p<0.001) and interleukin-6 (IL-6) levels (p=0.004), but not other classic inflammatory markers, were observed in obese patients compared to lean controls. Additionally, ProT-α levels were substantially higher in obese patients (median ProT-α, 600.0 vs 411.5 pg/mL, p=0.004), positively correlating with selected cytokines (TNF-α and IL-8). Notably, increments in circulating ProT-a were associated with increases in fasting insulin levels, suggesting a potential causal relationship. Hyperinsulinism, a compensatory mechanism for IR, drives cancer and cardiovascular risk in obese patients. Conclusions: This study represents the first investigation into ProT-α levels in human obesity, suggesting its potential utility as a sensitive biomarker of inflammation and IR in obese individuals, even before the onset of any cardiometabolic complications and changes in conventional inflammatory parameters. Further research is warranted to validate the role of this novel adipocyte-derived biomolecule in obesity-related pathophysiology.