Although arteriovenous fistula (AVF) is the recommended access for hemodialysis (HD), it carries a high risk for stenosis. Since osteopontin (OPN) is implicated in the process of vascular calcification in HD patients, OPN may be a marker for AVF stenosis. The present study evaluated OPN as a potential marker of AVF stenosis in HD patients. Diagnosing a stenotic lesion was made by combining B mode with color and pulse wave Doppler imaging. Criteria for diagnosis of stenotic AVF included 50% reduction in diameter in B mode in combination with a 2-3-fold increase of peak systolic velocity compared with the unaffected segment. The present study included 60 HD patients with stenotic AVF and 60 patients with functional AVF. Comparison between the two groups revealed that patients in the former group had significantly higher serum OPN levels [median (IQR): 17.1 (12.1-30.4) vs 5.8 (5.0-10.0) ng/mL, p<0.001]. All patients were classified into those with low (< median) and with high (≥ median) OPN levels. Comparison between these groups revealed that the former group had a significantly lower frequency of stenotic AVF (31.7 vs 68.3%, p<0.001) and a longer time to AVF stenosis [mean (95% CI): 68.4 (54.7-82.1) vs 46.5 (39.6-53.4) months, p=0.001]. OPN levels in HD patients may be useful markers for predicting and detecting AVF stenosis.