Microbiome-encoded β-glucuronidase (GUS) enzymes play important roles in human health by metabolizing drugs in the gastrointestinal (GI) tract. The numbers, types, and diversity of these proteins in the human GI microbiome, however, remain undefined. We present an atlas of GUS enzymes comprehensive for the Human Microbiome Project GI database. We identify 3,013 total and 279 unique microbiome-encoded GUS proteins clustered into six unique structural categories. We assign their taxonomy, assess cellular localization, reveal the inter-individual variability within the 139 individuals sampled, and discover 112 novel microbial GUS enzymes. A representative in vitro panel of the most common GUS proteins by read abundances highlights structural and functional variabilities within the family, including their differential processing of smaller glucuronides and larger carbohydrates. These data provide a sequencing-to-molecular roadmap for examining microbiome-encoded enzymes essential to human health. Display omitted •β-Glucuronidase (GUS) enzymes from the Human Microbiome Project examined•3,013 total and 279 unique microbial GUS proteins cluster in six structural groups•GUSs are universally present and exhibit distinct inter-individual variabilities•112 novel GUSs are discovered, and functional differentiation is established Microbial β-glucuronidase (GUS) enzymes cause drug-induced damage in the mammalian gut. We identify the 3,013 total and 279 unique microbial GUS proteins in the Human Microbiome Project database, and reveal their differential processing of distinct substrates. These data provide a roadmap for examining microbiome-encoded enzymes essential to human health.