Objective To assess whether an abnormality in cholesterol absorption or synthesis may be associated with hypocholesterolemia in patients with single ventricle anatomy following Fontan palliation. Study design This is a cross-sectional study of 21 patients with hypocholesterolemia following Fontan procedure and age/sex-matched healthy controls, with median age of 13.4 (IQR 10.6-16.1) years. Laboratory values of several biomarkers, including phytosterols and 5-α-cholestanol (for cholesterol absorption) and lathosterol (for cholesterol biosynthesis), as well as cholesterol levels, inflammatory markers, and indices of liver function were compared between patients following Fontan procedure and controls. Results The Fontan cohort had significantly lower total cholesterol (mean 117 ± SD 13.9, vs 128 ± 19.2 mg/dL, P =.03) and free cholesterol (35.5 ± 4.5 vs 39.2 ± 5.4 mg/dL, P =.02) compared with control patients. There was an increase in normalized 5-α-cholestanol (1.51 ± 0.6 vs 1.14 ± 0.37 μg/mL, P =.02), and a significantly lower lathosterol/5-α-cholestanol ratio (0.70 ± 0.38 vs 1.11 ± 0.76, P =.04). There was a strong correlation (r = 0.78, P <.0001) between lathosterol and cholesterol levels in the Fontan cohort, not seen in controls (r = 0.47, P =.04). The Fontan cohort also had significantly higher C-reactive protein, transaminases, total bilirubin, and gamma-glutamyl transferase levels. Conclusions Patients with hypocholesterolemia following Fontan procedure have evidence of increased cholesterol absorption and decreased cholesterol synthesis. As cholesterol absorption efficiency is a regulated process, this finding suggests an upregulation of cholesterol absorption as a result of decreased cholesterol production. In the setting of elevated liver indices and possible inflammation, this finding supports a growing body of data suggesting development of liver disease in patients receiving Fontan.