<p>The microbiome is widely involved in host metabolism, with many omics studies suggesting that it is important for metabolic health. Although studies in this area have made great strides in furthering our understanding of the role of the microbiome in health and disease, key challenges still hinder the safe clinical application of gut microbiota-targeted therapies. These limitations include a lack of confirmation of causality between the gut microbiota and host health, insights into the molecular mechanisms by which the gut microbiota functions to affect host health, and the development of therapeutic strategies that accurately regulate the function of the gut microbiota towards specific microbial enzyme targets without affecting its overall composition and viability. Microbial enzymes with various functions and activities have attracted the attention of many researchers in the past few years, especially microbiota–host isozymes, which are enzymes in the microbiome and the host that share a similar function. Such isozymes, as well as microbial-specific enzymes involved in basic biological processes of the gut microbiota, metabolism of nutrients, and synthesis of active metabolites and interactions in microbial-host communities, are the key mediators of gut microbiota–host crosstalk and have received much attention. In this Review, we provide a holistic understanding of the multifaceted role of gut microbial enzymes, including providing guidance for their discovery, while highlighting the great potential of gut microbial enzyme-oriented therapies for precision medicine.</p>