The significance of the gut microbiome in regulating host functions is tightly connected with host diet and an array of metabolites produced by diet-microbiome interactions. Sphingolipid and cholesterol are ubiquitous dietary constituents that are central to this metabolic relationship due to their constant exposure to gut microbes from infancy to adulthood. The roles of sphingolipid and cholesterol metabolism have been extensively studied in the context of host biology, but the role of these lipids in host-microbiome interactions is not well understood. Even in cases where a role for gut microbial-derived lipids in modifying host health has been demonstrated, the identity of the responsible microbes and resulting metabolites remain elusive. Each of the following chapters represents progression of work building on the theme of sphingolipid- and cholesterol- dependent microbiome-host interactions. In chapter 1, a bioorthogonal labeling based workflow is developed to identify sphinganine-specific gut bacterial interactors and the associated metabolites. In chapter 2, the above workflow is extended to elucidate cholesterol-interacting microbes, the downstream cholesterol-derivative, and how these interactions influence host cholesterol homeostasis. Chapter 3 summarizes the advantages and challenges of the workflow that utilized in the above chapters. In chapter 4, bioorthogonal labeling is utilized in another way to track bacterially-derived sphingolipid metabolites in host tissues, and the impacts on the host metabolism is also studied. Chapter 5 is the continuation of chapter 4 in which the roles of gut commensal sphingolipid production in modulating host skin biology is investigated. In chapter 6, we leverage the features of bioorthogonal labeling coupled with biochemical and computational tool to discover an uncharacterized enzymatic function in sphingolipid-producing bacteria. In chapter 7, we build upon the observation in chapter 1 by investigating the relationship between human milk sphingolipids and infant gut microbial composition in a longitudinal exclusively breast-fed infant cohort. Chapter 8 is a preliminary investigation of our active recruiting infant cohort study and shows the importance of intensive investigation to reveal sophisticated dynamics of microbial community configuration and metabolic capacity in the early-stage infant gut. Collectively, these chapters cover topics based on fundamental knowledge learned through model systems to its translation of this insight to human cohort studies. Overall, this work elucidates the specific microbial interactors with dietary lipids, the associated metabolites produced by these interactions and how these interactions influence diverse aspects of host physiology tying to lipid-dependent diet-microbiome-host interactions. The findings in this dissertation set a foundation for defining how bioactive lipids in the diet alter the function of the gut microbiome at a resolution that facilitates effective dietary interventions to improve host health.