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SCOPE: Poor vitamin D (vitD) status is linked to increased risk of infectious diseases, thus there is need for vitD‐rich foods. UVB‐exposed mushrooms synthesize vitD₂ but knowledge of bioavailability and function in immune response is lacking. METHODS AND RESULTS: One hundred rats were fed one of five diets—control, 20 IU vitD₃/day; no vitD₃/day; 5% unexposed mushroom, 2.4 IU vitD₂/day; 2.5% UVB mushroom, 300 IU vitD₂/day; and 5% UVB mushroom, 600 IU vitD₂/day—for 10 wk and challenged with either saline or the endotoxin LPS. Blood and tissues were collected at 3 h postchallenge. Plasma 25‐hydroxyvitamin D (25OHD) levels from UVB‐exposed mushroom fed rats were significantly elevated and associated with higher natural killer cell activity and reduced plasma inflammatory response to LPS compared to control diet fed rats. Microarray evaluation of rat spleens for changes in inflammatory gene expression showed significant upregulation of proinflammatory genes after LPS compared to saline controls in all groups. However, compared to control rats, upregulation of the proinflammatory genes was markedly reduced in the groups fed vitD₂‐enriched mushrooms. CONCLUSION: Rats fed UVB‐exposed mushrooms had significantly higher plasma total 25OHD levels that were associated with increased innate immune response and anti‐inflammatory effects.