High cholesterol synthesis at infancy could lead to hypercholesterolemia later in life. However, high synthesis at infancy could be traced to low dietary cholesterol especially in the formula-fed infants because they consume diets high in phytosterol (PS), a known cholesterol absorption inhibitor. High PS levels are found in vegetable oil used in infant foods. Human milk contains significant amounts of cholesterol ranging from 0.26-0.28mmol/L, compared to the very low levels in infant formula (IF) which can be as low as 0.08mmol/L. Therefore, the objective of this study was to investigate the effect of IF containing different levels of PS and cholesterol on circulating cholesterol levels, cholesterol absorption and synthesis, and other related health biomarkers using neonate piglets as model for human infants. A total of 32 piglets were used with 8 piglets per group fed diets of the following composition: (i) high in PS; low in cholesterol (HiPSLoChol), (ii) high in PS; high in cholesterol (HiPSHiChol), (iii) low in PS; high in cholesterol (LoPSHiChol) and (iv) low in PS; low in cholesterol (LoPSLoChol). After 21 days of study, various tissues were collected for analysis. In plasma and liver, cholesterol levels were higher p<0.05 in LoPSHiChol compared to LoPSLoChol diet group. Cholesterol synthesis levels in plasma, liver and distal intestine were also lower p<0.05 in the LoPSHiChol diet group compared to the HiPSLoChol and LoPSLoChol diet groups. Circulating lipid profiles after 3wks on each diet were compared across different dietary treatments. No significant differences were observed in circulating total cholesterol levels across the treatments. However, circulating levels of LDL-C in HiPSHiChol and LoPSHiChol increased (p<0.05) compared to the HiPSLoChol group. Plasma fatty acids shows higher (P<0.05) docosahexanoic acid (DHA) in HiPSLoChol diet compared to LoPSHiChol diet group. Also, ratio of n-3 to n-6 fatty acids was higher (P<0.05) in HiPSLoChol diet group compared to LoPSHiChol diet group. Beta and gamma tocopherols were lower in LoPSHiChol and LoPSLoChol diet groups compared to the remaining two groups. Other health biomarkers parameters measured such as antioxidant capacity, apolipoprotein A1 and B, C-reactive protein, insulin, glucagon-like peptide 1, fructosamine and LDL particle size were not significantly different across diets. In conclusion, lowering dietary PS with an increase in cholesterol level in commercial IF may support efficient absorption of dietary cholesterol, while maintaining low cholesterol synthesis levels. These results are anticipated to help manufacturing industries in proper formulation of infant food to achieve a closer dietary benefit found in infants consuming human milk.