The gut is the richest ecosystem of microbes in the human body and has great influence on our health. Despite many efforts, the set of microbes inhabiting this environment is not fully known, limiting our ability to identify microbial content and to research it. In this work, we combine new microbial metagenomic assembled genomes from 51,052 samples, with previously published genomes to produce a curated set of 241,118 genomes. Based on this set, we procure a new and improved human gut microbiome reference set of 3594 high quality species genomes, which successfully matches 83.65% validation samples’ reads. This improved reference set contains 310 novel species, including one that exists in 19% of validation samples. Overall, this study provides a gut microbial genome reference set that can serve as a valuable resource for further research. Here, Leviatan et al. produce 241,118 genome assemblies to produce a new human gut microbiome reference set of 3,594 species genomes, of which 310 represent previously undescribed species, making the catalog a valuable resource for further research.