Low serum 25-hydroxyvitamin D is a risk factor for osteoporosis, cardiovascular disease, diabetes, and cancer. Disruption of noncholesterol sterol absorption due to cholesterol-lowering therapies may result in reduced fat-soluble vitamin absorption. We have previously reported on the cholesterol-lowering efficacy and reduced sterol absorption of probiotic bile salt hydrolase active Lactobacillus reuteri NCIMB 30242; however, the effects on fat-soluble vitamins was previously unknown and the objective of the present study. DESIGN, SETTINGS, PATIENTS, AND INTERVENTION: The study was double-blind, placebo-controlled, randomized, parallel-arm, multicenter lasting 13 weeks. A total of 127 otherwise healthy hypercholesterolemic adults with low-density lipoprotein-cholesterol >3.4 mmol/L, triglycerides .05). L. reuteri NCIMB 30242 increased serum 25-hydroxyvitamin D by 14.9 nmol/L, or 25.5%, over the intervention period, which was a significant mean change relative to placebo of 17.1 nmol/L, or 22.4%, respectively (P = .003). To our knowledge, this is the first report of increased circulating 25-hydroxyvitamin D in response to oral probiotic supplementation.