Cholestatic jaundice is a potentially serious condition that requires early diagnosis for proper management. Fat-soluble vitamin (FSV) deficiency develops as a consequence of cholestasis. Vitamin D deficiency is common and remains a challenge in patients with cholestasis. Objectives of the study were to evaluate the serum 25-hydroxyvitamin D status in infants with cholestatic jaundice. This cross-sectional analytical study was conducted at department of Paediatric Gastroenterology and Nutrition of Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh, from January 2017 to June 2018 on purposively sampled infants. Infants who developed jaundice before three months of age, had direct bilirubin of more than 20.0% of the total bilirubin if total bilirubin is ≥5 mg/dl or more than 1.0 mg/dl if total bilirubin is <5 mg/dl, with pale stool and dark urine were included as cases while infants who visited hospital with a diagnosis of acute bronchiolitis, reactive airway disease and acute viral infection but no evidence of liver, gastrointestinal disease or renal disease checked by means of medical history, physical examination and a review of medical records were included as controls. Blood was collected from patients with cholestatic liver disease for liver biochemistries, prothrombin time, 25-hydroxyvitamin D and serum calcium. Blood was also collected from controls for 25-hydroxyvitamin D and serum calcium. 25-hydroxyvitamin D levels <15, 15 to 20 and >20 ng/ml were defined as vitamin D "deficiency", "insufficiency" and "sufficiency" respectively. Thirty patients and 30 controls were evaluated. Mean age in cases and controls were 113.43±74.08 and 145.50±88.62 days respectively (p=0.134). Biliary atresia was found to be the commonest cause, 18(60.0%), followed by idiopathic neonatal hepatitis (INH) 7(23.3%), choledochal cyst 4(13.3%) and 1 case of neonatal hepatitis (NH) due to CMV infection. The mean serum bilirubin (total) was 12.07±3.92 mg/dl, mean serum bilirubin (direct) 6.51±2.03 mg/dl, serum ALT 130.7±67.81 U/L, serum AST 135.07±52.54 U/L, prothrombin time 17.36±11.88 seconds, serum gamma-glutamyl transpeptidase (GGT) 700.3±555.89 U/L, alkaline phosphatase 560.37±283.12 U/L and serum albumin was 3.6±0.4 gm/dl. Mean serum calcium was 9.18±0.84 mg/dl. Mean 25-hydroxyvitamin D level in cholestatic patients was 14.7±5.75 ng/ml, compared to 27.68±10.44 ng/ml in controls (p=0.001). Vitamin D deficiency was found in 43.3% patients. The correlation between age at presentation and serum 25-hydroxyvitamin D levels was not significant (r = 0.051; p = 0.784). Statistically significant negative correlation (r = -0.389; p=0.034) was found between serum 25-hydroxyvitamin D and serum gamma-glutamyl transpeptidase. Serum calcium was found to have statistically significant positive correlation with 25-hydroxyvitamin D (r=0.692; p=0.001). Blood levels of 25-hydroxyvitamin D in patients with cholestasis were lower than those of controls. So, adequate vitamin D supplementation and monitoring in this population is of great importance.