Vitamin D deficiency and insufficiency are highly prevalent conditions worldwide due to several factors, including poor sun exposure. Shift workers may be exposed to the risk of hypovitaminosis D due to fewer opportunities for sunlight exposure compared to day workers. A systematic review of the PubMed, SCOPUS, and EMBASE databases was conducted according to the Preferred Reporting Items for Systemic Reviews and Meta-Analyses (PRISMA) statement to investigate the effect of shift work on vitamin D levels. Mean differences (MD) and 95% confidence intervals (CI) of serum 25-OH-D levels in shift workers and non-shift workers were calculated. A total of 13 cross-sectional studies were included in the meta-analysis. We found significantly lower levels of serum 25-OH-D in shift workers compared with non-shift workers (MD: −1.85, 95% CI [−2.49 to −1.21]). Heterogeneity among included studies was high (I2 = 89%, p < 0.0001), and neither subgroup analysis nor meta-regression were able to identify specific sources of the heterogeneity that may be related to the different characteristics of shift work among studies. The monitoring of serum vitamin D levels and prompt correction of any deficiencies should be considered in shift workers. Notably, since a large part of the observations are derived from Koreans, larger epidemiological studies are needed in other populations.