The fecal microbiota of two healthy adults was cultivated in a medium containing commercial fructooligosaccharides [FOS; 1-kestose (GF 2 ), nystose (GF 3 ), and 1 F -fructofuranosylnystose (GF 4 )]. Initially, the proportions of lactobacilli in the two feces samples were only 0.42% and 0.17%; however, they significantly increased to 7.2% and 4.8%, respectively, after cultivation on FOS. Most FOS-utilizing isolates could utilize only GF 2 ; however, Lacticaseibacillus paracasei strain Lp02 could fully consume GF 3 and GF 4 too. The FOS operon (fosRABCDXE) was present in Lc. paracasei Lp02 and another Lc. paracasei strain, KCTC 3510 T , but fosE was only partially present in the non-FOS-degrading strain KCTC 3510 T . In addition, the top six upregulated genes in the presence of FOS were fosABCDXE, particularly fosE. FosE is a β-fructosidase that hydrolyzes both sucrose and all three FOS. Finally, a genome-based analysis suggested that fosE is mainly observed in Lc. paracasei, and only 13.5% (61/452) of their reported genomes were confirmed to include it. In conclusion, FosE allows the utilization of FOS, including GF 3 and GF 4 as well as GF 2 , by some Lc. paracasei strains, suggesting that this species plays a pivotal role in FOS utilization in the human gut.