Reduced renal synthesis of 1,25-dihydroxyvitamin D (1,25(OH)2D) from 25-hydroxyvitamin D (25OHD) in end stage renal disease (ESRD) results in low serum 1,25(OH)2D. This appears to be due to reduced renal cell function and elevated serum fibroblast growth factor 23 (FGF23). Treatment strategies have therefore focussed on 1,25(OH)2D or its synthetic analogues, alfacalcidol or paricalcitol. However this overlooks 25-hydroxyvitamin D (25OHD) deficiency, which is common in ESRD. In the current study a subset of 33 haemodialysis patients from Coventry and Warwickshire were assessed during routine supplementation with vitamin D3 to raise serum 25OHD (colecalciferol supplementation: serum 25OHD <50 nmol/L repletion dose of 40,000IU for 3 months, ≥50 nmol/L maintenance dose of 20,000IU fortnightly, >150 nmol/L stop and recheck in 3 months). Multiple serum vitamin D metabolites were measured using liquid chromatography-tandem mass spectrometry at baseline (T0) and after 12 months supplementation (T12). Serum 25OHD increased significantly from 37.4±4.38 nmol/L at T0 to 117.7±6.61 nmol/L at T12 (P<0.001) with 88% of patients ≥75 nmol/L. Serum 1,25(OH)2D also increased significantly in 94% of patients (P<0.001) from 43.4±4.75 pmol/L to 91.2±5.30pmol/L at T12. Parallel analyses showed that serum calcium increased following colecalciferol supplementation (T0 vs. T12, 2.35±0.03 to 2.45±0.03 mmol/L, P<0.05), no hypercalcaemia was associated with colecalciferol supplementation. At T0 levels of 1,25(OH)2D and 24,25(OH)2D correlated with 25OHD (P<0.05). However, once 25OHD was replete (T12) this correlation was lost. Serum calcium correlated with 1,25(OH)2D at T12, but not at T0. These data suggest serum 25OHD was limiting at T0. This study indicates that vitamin D repletion in haemodialysis patients is safe and significantly increases serum 1,25(OH)2D. Demonstrating that kidneys with a low GFR and elevated FGF23 retain the ability to synthesise 1,25(OH)2D in a substrate-dependent fashion. Complementing 1,25(OH)2D analogue treatment with colecalciferol may prove effective in managing bone and mineral disorders associated with renal disease.