Objective To investigate the relation between 25-hydroxyvitamin D3[25(OH)D3] and coronary artery disease. Methods Three hundred and ten patients with selective coronary angiogram (CAG) were enrolled in this study and they were divided into two groups: non-coronary artery stenosis group with 76 patients and coronary artery stenosis group with 234 patients. The degree of coronary artery stenosis was evaluated by the international general Gensini integration system. The levels of fasting plasma glucose (FPG), total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL-C), low density lipoprotein (LDL-C) were detected by automatic biochemistry analyzer. The level of 25(OH)D3 was detected by tandem mass spectrometry. The relationship of Gensini integration scores and risk factors were analyzed. Logistic regression analysis was used in multicity factors analysis. Results The levels of age, Gensini integration scores, 25(OH)D3, FPG and LDL-C in non-coronary artery stenosis group and coronary artery stenosis group had significant differences (P<0.05). The number of coronary stenosis and Gensini integration scores in 25(OH) D3 deficiency group were significantly higher than those in non-25 (OH)D3 deficiency group (P<0.01). Logistic regression analysis showed that age, FPG and 25(OH)D3 levels were risk factors for coronary artery stenosis (P<0.01 or<0.05), and the level of 25(OH)D3 had negative correlation with coronary artery stenosis (B =- 0.100), and it was a protection factor (OR =0.904, 95%CI:0.911-0.983, P=0.000). Conclusions 25(OH)D3 deficiency is one of the risk factor of coronary artery disease.