Cobalamin (vitamin B12; VB₁₂) is an indispensable nutrient for all living entities in the Earth's biosphere and plays a vital role in both natural and host environments. Currently in the metagenomic era, gene families of interest are extracted and analyzed based on functional profiles by searching shotgun metagenomes against public databases. However, critical issues exist in applying public databases for specific processes such as VB₁₂ biosynthesis pathways. We developed a curated functional gene database termed VB₁₂Path for accurate metagenomic profiling of VB₁₂ biosynthesis gene families of microbial communities in complex environments. VB₁₂Path contains a total of 60 VB₁₂ synthesis gene families, 287,731 sequences, and 21,154 homology groups, and it aims to provide accurate functional and taxonomic profiles of VB₁₂ synthesis pathways for shotgun metagenomes and minimize false-positive assignments. VB₁₂Path was applied to characterize cobalamin biosynthesis gene families in human intestines and marine environments. The results demonstrated that ocean and human intestine had dramatically different VB₁₂ synthesis processes and that gene families belonging to salvage and remodeling pathway dominated human intestine but were lowest in the ocean ecosystem. VB₁₂Path is expected to be a useful tool to study cobalamin biosynthesis processes via shotgun metagenome sequencing in both environmental and human microbiome research. IMPORTANCE Vitamin B12 (VB₁₂) is an indispensable nutrient for all living entities in the world but can only be synthesized by a small subset of prokaryotes. Therefore, this small subset of prokaryotes controls ecosystem stability and host health to some extent. However, critical accuracy and comprehensiveness issues exist in applying public databases to profile VB₁₂ synthetic gene families and taxonomic groups in complex metagenomes. In this study, we developed a curated functional gene database termed VB₁₂Path for accurate metagenomic profiling of VB₁₂ communities in complex environments. VB₁₂Path is expected to serve as a valuable tool to uncover the hidden microbial communities producing this precious nutrient on Earth.