Objective: To test the "vitamin D-folate hypothesis for the evolution of human skin pigmentation."Methods: Total ozone mapping spectrometer (TOMS) satellite data were used to examine surface UV-irradiance in a large (n = 649) Australian crosssectional study population. Genetic analysis was used to score vitamin D-and folate-related gene polymorphisms (n = 22), along with two pigmentation gene variants (IRF4-rs12203592/HERC2-rs12913832). Red cell folate and vitamin D 3 were measured by immunoassay and HPLC, respectively.Results: i. Ultraviolet radiation (UVR) and pigmentation genes interact to modify blood vitamin levels; Light skin IRF4-TT genotype has greatest folate loss while light skin HERC2-GG genotype has greatest vitamin D 3 synthesis (reflected in both TOMS and seasonal data).ii. UV-wavelength exhibits a dose-response relationship in folate loss within light skin IRF4-TT genotype (305 > 310 > 324 > 380 nm). Significant vitamin D 3 photosynthesis only occurs within light skin HERC2-GG genotype, and is maximal at 305 nm.iii. Three dietary antioxidants (vitamins C, E, and β-carotene) interact with UVR and pigmentation genes preventing oxidative loss of labile reduced folate vitamers, with greatest benefit in light skin IRF4-TT subjects. The putative photosensitiser, riboflavin, did not sensitize red cell folate to UVR and actually afforded protection. iv. Four genes (5xSNPs) influenced blood vitamin levels when stratified by pigmentation genotype; MTHFR-rs1801133/rs1801131, TS-rs34489327, CYP24A-rs17216707, and VDR-ApaI-rs7975232.v. Lightest IRF4-TT/darkest HERC2-AA genotype combination (greatest folate loss/lowest vitamin D 3 synthesis) has 0% occurrence. The opposing,