Purpose Epidemiological studies have suggested that indoor hospital employees, either day or night shift workers, are at high risk of metabolic and cardiovascular diseases. Interestingly, previous reports have also described a higher prevalence of vitamin D (25OHD) deficiency among these workers. However, few studies have determined the monthly variations in 25OHD levels in indoor hospital employees. Methods To address this lack of knowledge, in 2018, during the periodic health surveillance checks at the Service of Occupational Medicine, we measured 25OHD levels in a group of indoor hospital workers (88 rotating night shift workers vs 200 day workers). Each participant received a single annual health surveillance check. Results The mean levels of 25OHD were consistently below the lower limit of the normal range in both groups throughout the year. Only in the summer, day workers but not rotating night shift workers (mean 25.9 ± 11.3 ng/ml vs 23.1 ± 9.1 ng/ml; p = 0.042) showed levels significantly higher than those in the other seasons. This difference remained statistically significant even after correction for study covariates [β = − 1.649 (CI − 0.283/− 3.482), p = 0.039]. A cosinor analysis confirmed that the difference in the 25OHD levels between groups was present later in the year. Conclusions We found that relatively young healthy hospital workers, especially those with rotating night shifts, in the absence of significant metabolic risk factors, have a high risk of 25OHD deficiency/insufficiency. Because 25OHD deficiency may lead to a progression to more severe conditions such as osteoporosis or bone fractures, our results should be verified in larger cohorts including different ancestries.