Serum 1,25(OH)2D and 24,25(OH)2D are decreased in CKD. Megalin in proximal tubular epithelial cells reabsorbs glomerular-filtered 25(OH)D-DBP complex to convert 25(OH)D to 1,25(OH)2D and 24,25(OH)2D. Urinary C-megalin excretion is increased via exocytosis from injured nephrons overloaded with megalin-mediated protein metabolism. This study investigated the significance of urinary C-megalin excretion in vitamin D metabolism in 153 pre-dialysis CKD patients. Urinary C-megalin was positively associated with urinary protein, β2MG and α1MG, and exhibited negative correlations with serum 25(OH)D, 1,25(OH)2D and 24,25(OH)2D. Multiple regression analysis showed that urinary C-megalin had a significantly negative association with 25(OH)D. Serum 1,25(OH)2D and 24,25(OH)2D, as well as 1,25(OH)2D/25(OH)D and 24,25(OH)2D/25(OH)D ratios, showed positive correlations with eGFR. Additionally, wholePTH was positively associated with 1,25(OH)2D/25(OH)D and 1,25(OH)2D/24,25(OH)2D, while FGF23 was positively associated with 24,25(OH)2D/25(OH)D and negatively with 1,25(OH)2D/24,25(OH)2D. Urinary C-megalin emerged as an independent factor positively associated with 1,25(OH)2D/25(OH)D and 1,25(OH)2D/24,25(OH)2D. Although 1,25(OH)2D and 24,25(OH)2D are decreased in CKD patient serum, our findings suggest that PTH and FGF23 retain their effects to regulate vitamin D metabolism even in the kidneys of these patients, while production of 1,25(OH)2D and 24,25(OH)2D from 25(OH)D is restricted due to either impairment of megalin-mediated reabsorption of the 25(OH)D-DBP complex or reduced renal mass.