This study establishes an advanced reference genome collection for giant panda gut microbiota through integrated culturomics and long-read sequencing. Analysis of 142 biological specimens—including fecal samples from healthy captive pandas and digestive tract contents from deceased individuals—enabled comprehensive microbial characterization. Culturomics yielded 76 isolated bacterial strains representing 62 species, with three novel phylogenetic lineages identified. Parallel PacBio HiFi sequencing generated 44.45 million high-accuracy reads, producing 183 non-redundant metagenome-assembled genomes. Sixty-nine genomes exceeded stringent quality thresholds (>99% completeness, <3% contamination).

Integration of these methodologies expanded the PBAC database to 466 species-level genomes, an 84% increase from the initial version. The updated resource contains 213 newly documented microbial species and five novel genera. Validation confirms 92.7% metagenomic and 91.9% metatranscriptomic mapping efficiency for core genomes. The fifty most abundant references capture 89% of sequencing data, predominantly derived from project-specific isolates and assemblies. This genomic resource significantly advances research on bacterial adaptations in bamboo-digesting ecosystems and supports conservation efforts for endangered ursids.