Background Prolyl carboxypeptidase (PRCP) is involved in the regulation of body weight, likely by hydro-lysing alpha-melanocyte-stimulating hormone and apelin in the hypothalamus and in the periphery. A link between PRCP protein concentrations in plasma and metabolic disorders has been reported. In this study, we investigated the distribution of circulating PRCP activity and assessed its relation with body weight and adipose tissue in obese patients and patients who significantly lost weight. Methods PRCP activity was measured using reversed-phase high-performance liquid chromatography in different isolated blood fractions and primary human cells to investigate the distribution of circulating PRCP. PRCP activity was measured in serum of individuals (n = 75) categorized based on their body mass index (BMI < 25.0; 25.0–29.9; 30.0–39.9; 40.0 kg/ m2) and the diagnosis of metabolic syndrome. Differences in serum PRCP activity were determined before and six months after weight loss, either by diet (n = 45) or by bariatric surgery (n = 24). Potential correlations between serum PRCP activity and several metabolic and biochemical parameters were assessed. Additionally, plasma PRCP concentrations were quantified using a sensitive ELISA in the bariatric surgery group. Results White blood cells and plasma contributed the most to circulating PRCP activity. Serum PRCP activity in lean subjects was 0.83 ± 0.04 U/L and increased significantly with a rising BMI (p<0.001) and decreased upon weight loss (diet, p<0.05; bariatric surgery, p<0.001). The serum PRCP activity alteration reflected body weight changes and was found to be positively correlated with several metabolic parameters, including: total, abdominal and visceral adipose tissue. Plasma PRCP concentration was found to be significantly correlated to serum PRCP activity (0.865; p<0.001). Additionally, a significant decrease (p<0.001) in plasma PRCP protein concentration (mean ± SD) before (18.2 ± 3.7 ng/mL) and 6 months after bariatric surgery (15.7 ± 2.7 ng/mL) was found. Conclusion Our novel findings demonstrate that white blood cells and plasma contributed the most to circulating PRCP activity. Additionally, we have shown that there were significant correlations between serum PRCP activity and various metabolic parameters, and that plasma PRCP concentration was significantly correlated to serum PRCP activity. These novel findings on PRCP activity in serum support further investigation of its in vivo role and involvement in several metabolic diseases.