This study aimed to examine the association between rotating night shift work and carotid atherosclerosis in Chinese steelworkers. A total of 3582 subjects were included in this study. Different exposure metrics of night shifts, including current shift status, duration of night shifts (years), the cumulative number of night shifts (nights), cumulative length of night shifts (hours) and average frequency of night shifts (nights/month), were used to examine the relationship between night shift work and carotid atherosclerosis, including carotid plaque and carotid plaque scores. The prevalence of carotid plaque was 29.9% in the study population. Compared with day workers, significantly increased odds of carotid plaque were observed among shift workers in each quartile of the duration of night shifts after adjusting for potential confounders, especially in the third and fourth quartiles: odds ratio (OR) = 1.91, 95% confidence interval (CI): 1.46 to 2.49, in the third quartiles; OR = 1.69, 95% CI: 1.30 to 2.20, in the fourth quartiles. Positive associations of different exposure metrics of night shifts (continuous) with the odds of carotid plaque and the carotid plaque score were observed in the restricted cubic spline (RCS) models. No significant association was found in female workers between night shift work and carotid plaque. Rotating night shift work is associated with elevated odds of carotid atherosclerosis in male steelworkers.