Unusual bile acids (1β-hydroxylated bile acids), particularly 1β-hydroxyl-cholic acid (CA-1β-ol) and 1β-hydroxyl-chenodeoxycholic acid (CDCA-1β-ol), have been detected in the urine of infants. These acids are conjugated with amino acids, such as taurine, and are then excreted mainly via the urine. CA-1β-ol and CDCA-1β-ol are the predominant bile acids during infancy and are present in relatively large amounts in the urine. However, the biosynthetic pathway of 1β-hydroxylated bile acids in infants remains unclear. To investigate the biosynthetic pathway of 1β-hydroxylated bile acids during infancy, we performed a metabolic reaction using infant hepatocytes at 3 months after delivery. Glyco- and tauro-CA-1β-ol were identified by LC/tandem mass spectrometry (MS/MS) analysis of the extracted culture medium incubated with cholic acids (CAs). Further, we identified that ketoconazole suppressed CA 1β-hydroxylation and that the CYP3A subfamily was the primary group of enzymes responsible for CA-1β-ol formation. The present study provides new information about the biosynthetic pathway of 1β-hydroxylated bile acids during infancy.