Impaired wound healing is an important problem in diabetes mellitus; however, its pathogenesis remains unclear. We aimed to evaluate serum prolidase activity (SPA), an important marker of collagen turnover, in subjects with and without diabetic foot ulcers as compared with healthy controls. Twenty-seven patients with diabetic foot ulcers (foot ulcer group), 27 patients without diabetic foot ulcers (diabetic control group) and 27 healthy controls were enrolled. The study groups had similar age, sex distribution and body mass index. Metabolic and inflammatory parameters as well as SPA were determined. The diabetic foot ulcer group had significantly higher SPA (both p < 0.001) when compared with the diabetic and the healthy control groups. SPA showed a positive correlation with high-sensitive C-reactive protein and a negative correlation with high-density lipoprotein cholesterol levels (r = 0.313, p = 0.021 and r = -0.233, p = 0.036, respectively). No correlation was detected between SPA and glycaemic parameters. SPA appears to be higher in patients with diabetic foot ulcers when compared with patients without diabetic foot ulcers and healthy controls. The underlying mechanisms of elevated SPA and its clinical significance in predicting the natural course of wound healing in diabetic foot ulcers needs to be further evaluation.