Aim To observe the relationship between protein kinase C (PKC) activity and anterior tibial artery calcification score in patients with diabetic foot. Methods 60 diabetic patients hospitalized for above-knee amputation in the Department of Orthopedics, Affiliated Hospital of Jiangsu University were recruited from October 2012 to January 2016. The patients were categorized based on the severity of anterior tibial artery calcification, which was assessed by computed tomography (CT), into mild calcification group (0＜calcification score＜100, n＝ 20), moderate stenosis group (100≤calcification score＜200, n＝20) and severe calcification group (calcification score≥200, n＝20). Baseline clinical data regarding sex, age, duration of diabetes mellitus, hypertension status, fasting plasma glucose (FPG), glycated hemoglobin (HbA1c), and lipid profile were collected before the amputation. Von kossa staining, PepTag Assay and Western blot were then performed. Results Similarly with Von Kossa staining, CT scan results showed that calcium deposition in anterior tibial artery plaques of diabetic patients hospitalized for above-knee amputation were enlarged and calcium particles density in moderate and severe groups were stronger than that in mild group. The calcification in all calcification groups were showed as spotty calcification. PepTag Assay, Western blot and Pearson correlation showed that anterior tibial artery calcification score in diabetic patients hospitalized for above-knee amputation was positively associated with PKC activity (r＝0.931, P＜0.001). Further analysis indicated that alpha subtypes of PKC maybe played an important role to promote the evolution of calcification. Conclusion The activation of PKC subtypes may be related with the evolution of calcification in atherosclerotic plaques, so as to further target the mechanism of vascular calcification.