Human milk oligosaccharides (HMOs) have garnered significant attention as one of the bioactive components in human milk, with growing applications in infant formula and food products. HMOs enhance butyrate production, which is produced by butyrate-producing bacteria such as Faecalibacterium prausnitzii and contributes to gut health through its diverse biological functions. However, the specific mechanisms by which individual HMOs promote butyrate production remain unclear. In this study, we conducted in vitro co-culture experiments of F. prausnitzii and Bifidobacterium bifidum, examining their relative abundance, fatty acid production, residual sugar levels, and gene expression. Our results revealed that B. bifidum utilizes HMOs and provides the constituent sugars to F. prausnitzii, thereby promoting butyrate production by F. prausnitzii. Furthermore, we found that the underlying mechanisms vary depending on the structure of the HMOs. Specifically, 2′-fucosyllactose and 3′-sialyllactose enhance the butyrate production efficiency of F. prausnitzii, while 6′-sialyllactose primarily promotes the growth of F. prausnitzii. These findings not only deepen our understanding of how HMOs influence infant gut health but also suggest new directions for developing nutritional products that leverage the distinct functional properties of each HMO.