Optimal vitamin D status has been suggested to increase treatment success of women undergoing assisted reproductive treatments. However, inconclusive data raises the need for more comprehensive studies. This study investigates the relationship between the levels of vitamin D metabolites in follicular fluid and outcome of IVF/ICSI treatment. The levels of 25OHD3, 1,25(OH)2D3 and 24,25(OH)2D3 were measured in follicular fluid and serum samples from 116 women undergoing IVF/ICSI treatment and expression of the vitamin D regulating enzymes (CYP2R1, CYP27B1, CYP24A1) and the vitamin D receptor (VDR) were investigated in human ovaries. The vitamin D metabolites 25OHD3, 1,25(OH)2D3 and 24,25(OH)2D3 were present in follicular fluid and in serum. 25OHD3 levels were similar, while levels of 24,25(OH)2D3 were higher and 1,25(OH)2D3 lower in follicular fluid than in serum. Local vitamin D metabolism is supported by CYP2R1, CYP27B1, CYP24A1 and VDR expression in the follicle. When stratifying women according to live birth outcome, women achieving a live birth had significantly higher follicular fluid concentration of the 24,25(OH)2D3 metabolite (17.4 ± 8.5 versus 13.5 ± 6.3 nmol/L, p = 0.015) and a higher 24,25(OH)2D3/25OHD3 ratio (0.25 ± 0.07 versus 0.21 ± 0.07, p = 0.029) compared with women with an unsuccessful cycle. Moreover, when grouping women according to their follicular fluid 24,25(OH)2D3/25OHD3 ratio (<15%, 15-85% and >85%) the live birth rate significantly increased across increasing ratio intervals (6%, 21% and 41%, respectively, p = 0.043). This study suggests that a higher follicular fluid 24,25(OH)2D3 level and 24,25(OH)2D3/25OHD3 ratio is associated with increased live birth rate in women undergoing IVF/ICSI treatment indicating that local vitamin D metabolism in the ovarian follicle may affect IVF/ICSI outcome.