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Background: Vitamin D deficiency became increasingly common worldwide and was related to various disorders. However, there was little information concerning the relationship between serum 25-hydroxyvitamin D (25(OH) D) level and shift work. The aim of this paper was to investigate the relation between serum 25(OH) D level and shift work in the American citizens. Methods: From the 2005–2006 NHANES, a total of 3025 participants were included. The relation between serum 25(OH) D level and shift work was investigated through the regression models. Results: There was an inverse relationship between serum 25(OH) D level and female shift worker in the fully adjusted models (β coefficient = −8.774, 95% confidence intervals [CI] = −16.83–−0.71, P = 0.03). Additionally, a negative correlation was presented between serum 25OHD level and Mexican American (β coefficient = −9.825, 95% CI = −19.31–−0.33, P = 0.04). No significant differences in serum 25(OH) D level were presented among indoor/outdoor works or manual/nonmanual works. However, long working hours (>40 h/week) were remarkably correlated with low serum 25(OH) D level (β coefficient = −8.007, 95% CI = −15.32–−0.68, P = 0.03). Conclusions: Our study concluded there was an inverse correlation between serum 25(OH) D level and shift work, especially in female population.