Objective To investigate the changes of plasma amino acid spectrum and its clinical significance in infants with CBAS2(congenital bile acid synthesis disorder type 2 ). Methods The clinical data of children with infantile cholestasis treated from January 2016 to June 2021 were retrospectively analyzed. According to the etiology, they were divided into CBAS2 group, neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD ) group and idiopathic neonatal intrahepatic hepatitis (INH) group. The differences of 22 amino acids in plasma among the three groups were compared. Results A total of 85 infantile cholestasis patients (58 boys and 27 girls) were included, and the median age was 2.3 (2.0-4.0) months. The median total bilirubin was 134.9 (103.1-181.7) μmol/L, and the median direct bilirubin was 79.0 (62.2-110.6) μmol/L. Among the 12 patients in CBAS2 group, 3 or more amino acids changed in 11 patients (91.7%) and branched chain amino acid (leucine, isoleucine and valine) was normal in 11 patients (91.7%). In the INH group, 17 (73.9%) of the 23 patients had≥3 amino acid changes. All the 50 patients in NICCD group had ≥3 amino acid changes. Compared with INH group, the levels of asparagine, serine, threonine and tyrosine in CBAS2 and NICCD groups were higher. Compared with NICCD group, the levels of glutamine, alanine, tryptophan, leucine and alanine/citrulline were higher, while the levels of citrulline, arginine, methionine, basic amino acid and threonine/serine were lower in CBAS2 group, and the differences were statistically significant (P<0.05). Conclusions Plasma amino acid spectra in children with CBAS2 vary widely. The changes of plasma amino acid spectra in CBAS2 children are different from those in NICCD and INH children, which is of great significance for the diagnosis and differential diagnosis of CBAS2.