Introduction: Despite the vulnerability of airline pilots (APs) to sleep disturbances, the biological underpinnings responsible for this phenomenon are still not entirely elucidated. However, there is an increasing amount of evidence indicating an association between 25-hydroxyvitamin D, Ca 2+ , and Mg 2+ levels and sleep health. In this cross-sectional study, we sought to examine the potential associations between serum levels of these biomarkers and the occurrence of poor sleep among APs.Methods: We examined a convenience sample of 100 male APs who underwent the Pittsburgh Sleep Quality Index (PSQI) to assess their sleep quality. Those who scored 5 or higher on the PSQI were labeled as poor sleepers. Serum levels of 25-hydroxyvitamin D, Ca 2+ , and Mg 2+ were quantified in all participants.Results: Out of the 100 APs, 58 (58%) and 42 (42%) were classified as good and poor sleepers, respectively, based on the PSQI scores. We defined vitamin D deficiency as serum 25-hydroxyvitamin D levels below 10 ng/mL and insufficiency as levels ranging from 10 to 30 ng/mL. The results revealed no significant differences in serum levels of 25-hydroxyvitamin D between the two groups, and there was no evidence of vitamin D deficiency or insufficiency. However, poor sleepers exhibited significantly lower levels of both Mg 2+ (1.8 ± 0.1 mg/dL versus 2.0 ± 0.1 mg/dL, respectively) and Ca 2+ (8.5 ± 0.4 mg/dL versus 9.1 ± 0.5 mg/dL, respectively) compared to good sleepers (P < 0.001 for both). Logistic regression analysis identified both Mg 2+ and Ca 2+ as independent biomarkers associated with poor sleep quality in APs ( P < 0.001 for both).Conclusion: Lower serum concentrations of Mg 2+ and Ca 2+ , not 25-hydroxyvitamin D, may be associated with poor sleep in APs.