Objective To describe serum 25(OH)D changes after Roux-en-Y gastric bypass (RYGB) and to determine if fat mass (FM) loss and vitamin D intake are associated with changes in serum levels. Design and Methods: The relationship between serum 25(OH)D and 1) FM, 2) weight, 3) % excess weight loss (EWL), and 4) BMI was investigated after controlling for potential confounders using a mixed effects linear model in 20 women before and up to 1-year post-RYGB. Subcutaneous (SAT) and visceral adipose tissue (VAT) vitamin D concentrations at time of RYGB were also evaluated. Results Weight and FM decreased 1-year after surgery by 45 ± 1 kg and 37 ± 1 kg, respectively while 25(OH)D increased by 10 ± 2 ng mL-1. Weight, FM, BMI, and %EWL changes were associated with 25(OH)D change. VAT had an average 21% more vitamin D per gram than SAT and concentrations were highly correlated. Conclusions Although weight loss may lead to increased serum 25(OH)D after RYGB, low levels remain a concern in some patients 1-year postsurgery. Additional research is needed to clarify the relationship between adipose storage of vitamin D and serum 25(OH)D in obesity, and how that relationship might change after surgery. This could lead to improved clinical management of vitamin D in this ever-growing clinical population.