Perturbed serum vitamin D level has been demonstrated to be associated with increased risk of cardiovascular disease. This study aimed to investigate the association of serum 25‐hydroxyvitamin D3 [25(OH)D3] levels with B ultrasonography‐detected carotid plaque and carotid intima‐media thickness (C‐IMT) in Chinese middle‐aged and elderly men. A total of 1001 men, aged 45‐78 years, were enrolled. Increased C‐IMT was defined as any C‐IMT value in the highest quartile of the study subjects (≥ 0.75 mm). The study population had a median serum 25(OH)D3 level of 14.51 ng/mL (interquartile range: 10.84‐18.67). Subjects with carotid plaque had lower serum 25(OH)D3 levels than those without [13.80 ng/mL (10.82‐17.68) vs. 14.74 ng/mL (10.87–19.08), P = 0.029], and decreasing serum 25(OH)D3 levels were accompanied by increased C‐IMT in both groups [with plaque: 13.24 ng/mL (9.91–16.81) vs. 14.45 ng/mL (11.40–18.51), P < 0.05; without plaque: 13.90 ng/mL (9.99‐17.09) vs. 14.99 ng/mL (11.17‐19.43), P < 0.01]. Multivariate logistic regression analysis showed that serum 25(OH)D3 levels were independently associated with carotid plaque [OR (95% CI): 0.972 (0.946‐0.998), P = 0.032)]. Additionally, serum 25(OH)D3 level was identified as an independent protective factor for increased C‐IMT among subjects with plaque [OR (95% CI): 0.900 (0.849‐0.955), P = 0.001] as well as those without [OR (95% CI): 0.944 (0.908‐0.981), P = 0.004]. These findings collectively suggest that serum 25(OH)D3 levels were inversely associated with atherosclerosis in Chinese middle‐aged and elderly men. This article is protected by copyright. All rights reserved.