As a dominant member of individuals’ gut microbiomes especially in non-industrialized populations, Segatella has been linked to various health outcomes of the host. However, these reported correlations were often conflicting, leading to debates regarding the specific role Segatella plays within the host intestinal environment. Taxonomic analysis based on metagenomic assembly and genome isolation shed light on the genetic diversity of Segatella, providing potential explanations for these conflicting roles. However, the limited availability of isolates has restricted our understanding of how members’ genetic diversity translates into phenotypic diversity. Those include the microbiota-derived metabolites, an important component of the gut microbiome mediating the host-microbe and inter-microbial communications. In this study, we have obtained 63 isolates from diverse lineages of Segatella within the confines of a single gut microbiome. Genomic and phylogenetic analyses revealed that these isolates can be clustered into six distinct clades which exhibit vastly different gene contents. Secondly, we performed comparative analyses that exposed differences in cellular morphologies and antimicrobial susceptibilities, as well as differential production of important bioactive molecules with reported associations with host health. For example, short-chain fatty acids (SCFAs) yielded from their distinct preferences for polysaccharide utilization. Strain sphingolipidomes were also investigated after identifying the key enzymes involved in sphingolipid synthesis, revealing distinct sphingolipid profiles of Segatella isolates that suggest different roles in intestinal interactions and communication. We further showed that exposure to Segatella lineages evokes varying transcriptional responses in human intestinal epithelial cells. Additionally, competition and cross-feeding behaviors were observed between different Segatella clades, aligning with their distinct metabolic profiles. Characterization of the microbial metabolism enhanced our understanding of how genomic variations drive phenotypical differences, affecting bacterial behaviors and host-microbe interactions. Our work uncovered significant phenotypic differences within related Segatella isolates, providing insights into their roles in the human intestinal environment and contributing to resolving this complex scientific puzzle.