Background: The type of feeding can differently influence bile acid ontogeny during the period of physiologic cholestasis that characterizes early human development. Aim: To investigate the effects of feeding from birth, conventional or partially protein hydrolyzed cow’s milk formulas were compared with breast milk regarding bile acid concentration and the composition of gallbladder bile in newborn guinea pigs. Methods: Forty newborn guinea pigs were allocated to one of three different feeding regimens: breast milk (n = 14), intact protein formula (n = 13) and partially protein hydrolyzed formula (n = 13). After 8 days, one third of the pups from each group was sacrificed; another third was sacrificed on the 14th day and the remainder on the 20th day of life. Bile acids in gallbladder bile were analyzed by gas chromatography/mass spectrometry and HPLC. Results: During the first 2 weeks of life, weight gain was significantly higher in breast-fed than in artificially fed pups. An age-related increase in total biliary bile acid concentrations was evident for breast-fed and hydrolyzed formula-fed animals, but not for those fed intact protein formula. Breast-fed animals had the highest total biliary bile acid concentrations on day 20 of life, with significant increases in chenodeoxycholic and 7-oxo-lithocholic acid concentrations, which were absent in the other two groups. Concentrations of 7-oxo-lithocholic acid on day 8 were significantly higher in animals fed intact protein formula compared with breast-fed and partially protein hydrolyzed formula-fed animals. Conclusions: In newborn guinea pigs, breast feeding is associated with a marked increase in biliary bile acid concentrations, which was not observed in artificially fed animals. The higher biliary bile acid concentrations and better weight gain in our breast-fed animals may reflect a greater feed efficiency associated with natural feeding. Biliary bile acid composition on day 8 suggests more rapid intestinal bacterial bile acid biotransformation in animals fed intact protein formula compared to other feeding regimens.