Context: Although studies have linked Vitamin D deficiency with immune-mediated diseases, data demonstrating a direct effect on T α-Cell function are sparse. Objective: Our objective was to determine whether oral Vitamin D3 influences T α-Cell activation in humans with Vitamin D deficiency. Design: This was a single-center ancillary study within VitaminDTherapy in Individuals at High Risk of Hypertension, a double-blind, multicenter, randomized controlled trial. Setting: This study was undertaken in a single academic medical center. Participants: Adults with vitaminDdeficiencyanduntreated pre-or early stage I hypertensionwere included. Intervention: In Vitamin D Therapy in Individuals at High Risk of Hypertension, participants were randomized to either low-(400 IU daily) or high-(4000 IU daily) dose oral Vitamin D3 for 6 months. In this ancillary study of 38 patients, we measured CD4 T α-Cell activation estimated by intracellular ATP release after stimulation of whole blood with plant lectin phytohemagglutinin collected at baseline (pretreatment) and 2-month follow-up. Main Outcome Measure: Determining whether ATP level changes were significantly different between treatment groups was the main outcome measure. Results: Treatment with 4000 IU of Vitamin D3 decreased intracellular CD4ATP release by 95.5 ng/ml (interquartile range, 219.5 to 105.8). In contrast, 400 IU of Vitamin D3 decreased intracellular CD4 ATP release by 0.5 ng/ml (interquartile range,69.2 to 148.5). In a proportional odds model, high-dose Vitamin D3 was more likely than low-dose Vitamin D3 to decrease CD4ATP release (odds ratio, 3.43; 95% confidence interval, 1.06-1.11). Conclusions: In this ancillary study of a randomized controlled trial, we found that high-dose Vitamin D3 significantly reducedCD4T α-Cell activation compared to low-dose Vitamin D3, providinghumanevidence that Vitamin D can influence cell-mediated immunity.