Background: Normal profiles of FBAs in healthy neonates and children in Kunming city and surrounding areas in China have not been previously determined. The objective of this study was to determine a developmental pattern of fecal bile acids (FBAs) in healthy neonates and children. Material/Methods: A cross-sectional study was performed on 238 healthy neonates and children recruited in the First Affiliated Hospital of Kunming Medical University, China from October 2015 to September 2016. Secreted primary and secondary FBAs in fresh feces were quantitated by liquid chromatography mass spectrometry (LC-MS). Amounts of FBAs in feces were compared among various age groups. Results: Trace amounts of cholic acid and chenodiol acid of primary FBAs were detectable at day 3 after birth, with a significant increase from day 3 to day 7. The primary FBAs gradually decreased from day 25 to the age of 6 years old. In contrast, a significant amount of glycochenodeoxycholic acid was detected on day 3 but decreased to a trace amount by day 7 and onwards. Primary FBAs appeared to maintain a high level, accounting for 98% of total FBAs, with no significant changes from day 7 to day 25 after birth. They gradually decreased from 90% to 10% from age 6 months to 6 years old. While the secondary FBAs were barely detected in neonates, only accounting for 2% of total FBAs, they were gradually elevated to 90% of total FBAs from age 6 months to 6 years old. Conclusions: The liver can effectively synthesize primary bile acids 7 days after birth, and fecal primary bile acids tend to be stable after the neonate stage. Secondary bile acids continuously increase along with the maturation of intestinal flora, which reaches a relatively stable level at around 3 years old. © Med Sci Monit, 2021; 27: e928214