Vitamin D is an essential micronutrient required for normal physiological function and recognized for its role regulating calcium metabolism. Recent work is beginning to emerge demonstrating a role for vitamin D in chronic illnesses, such as cancer. Circulating serum levels of 25(OH)D_2/3 were quantitatively measured using sensitive ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) in 406 lung cancer cases and 437 population controls, while 1,25(OH)₂ D_2/3 levels were measured in a subset of 90 cases and 104 controls using the same method, from the NCI-MD case-control cohort. 25(OH)D₃ levels were inversely associated with lung cancer status across quartiles (Q2 vs. Q1: OR_adjusted = 0.5, 95% CI = 0.3-0.8; Q3 vs. Q1: OR_adjusted = 0.5, 95% CI = 0.3-0.8; Q4 vs. Q1: OR_adjusted = 0.5, 95% CI = 0.2-0.9; P_trend = 0.004). Levels of 1,25(OH)₂ D₃ were also inversely associated with lung cancer status (Q2 vs. Q1: OR_adjusted = 0.2, 95% CI = 0.03-0.7; Q3 vs. Q1: OR_adjusted = 0.1, 95% CI = 0.01-0.4; Q4 vs. Q1: OR_adjusted = 0.04, 95% CI = 0.01-0.3; P_trend <0.0001). Although the observed trends were similar for the 25(OH)D₂ (P_trend = 0.08), no significant associations were seen between vitamin D₂ and lung cancer status. Additionally, genotyping of 296 SNPs in the same subjects resulted in findings that 27 SNPs, predominantly in CYP24A1 and VDR genes, were significantly associated with lung cancer status, affected mRNA expression, and modulated vitamin D levels. These findings suggest a protective role for vitamin D₃ in lung cancer, with similar trends but insignificant findings for D₂ . Vitamin D₃ levels appeared to be modulated by genetic variation in CYP24A1 and VDR genes. Additional research to illuminate the mechanism(s) through which vitamin D exacerbates effects against lung carcinogenesis is warranted.