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Background: Subclinical atherosclerosis is the asymptomatic phase of carotid atherosclerosis, and its early diagnosis is important to prevent cerebrovascular diseases. Although the vitamin D plays a role in the structure of vessels, the association between the serum level of vitamin D and subclinical atherosclerosis has not been well-studied. We aimed to investigate the association between serum vitamin D level and carotid artery intima-media thickness (CIMT) in Iranian population. Methods: One hundred individuals with the age range from 20 to 50 years with no history of cardiovascular risk factors were selected for the analysis. Measurements of serum 25-hydroxyvitamin D3 [25(OH) D3] concentration and CIMT were made. Confounding factors such as diabetes, hypertension (HTN), smoking, alcohol, tobacco, dyslipidemia, cardiovascular disease (CVD), high body mass index (BMI), history of drug intake especially calcium, vitamin D, statins, and anti-hypertensive drugs were considered and then excluded from our study. Results: The mean serum vitamin D level was  15.55 ± 0.42 ng/ml, whereas in the increased intima-media thickness (IMT), it was 12.50 ± 9.50 ng/ml. 55% of the subjects were diagnosed with subclinical atherosclerosis (IMT ≥ 0.75 mm). Mean IMT was  0.74 ± 0.12 mm; however, it was higher (0.86 ± 0.30) in severe vitamin D deficiency group. The analysis showed an association between serum 25(OH) D3 level and CIMT (P = 0.002). 44% of those participants with subclinical atherosclerosis had also a severe vitamin D deficiency, while only 13% of normal people had a severe vitamin D deficiency. Also, a correlation was observed between severe vitamin D deficiency and the presence of plaque or higher IMT. Conclusion: Serum 25(OH) D3 level was inversely correlated with CIMT in our investigated subjects with no cardiovascular risk factor.