BACKGROUND: The imbalanced respiratory microbiota observed in pneumonia causes high morbidity and mortality in childhood. Respiratory metagenomic analysis demands a comprehensive microbial gene catalogue, which will significantly advance our understanding of host-microorganism interactions. RESULTS: We collected 334 respiratory microbial samples from 171 healthy children and 76 children with pneumonia. The respiratory microbial gene catalogue we established comprised 2.25 million non-redundant microbial genes, covering 90.52% of prevalent genes. The major oropharyngeal microbial species found in healthy children were Prevotella and Streptococcus. In children with Mycoplasma pneumoniae pneumonia (MPP), oropharyngeal microbial diversity and associated gene numbers decreased compared with those of healthy children. The concurrence network of oropharyngeal microorganisms in patients predominantly featured Staphylococcus spp. and M. pneumoniae. Functional orthologues, which are associated with the metabolism of various lipids, membrane transport, and signal transduction, accumulated in the oropharyngeal microbiome of children with pneumonia. Several antibiotic resistance genes and virulence factor genes were identified in the genomes of M. pneumoniae and 13 other microorganisms reconstructed via metagenomic data. Although the common macrolide/β-lactam resistance genes were not identified in the assembled M. pneumoniae genome, a single-nucleotide polymorphism (A2063G) related to macrolide resistance was identified in a 23S ribosomal RNA gene. CONCLUSIONS: The results of this study will facilitate exploration of unknown microbial components and host-microorganism interactions in studies of the respiratory microbiome. They will also yield further insights into the microbial aetiology of MPP. © The Author(s) 2019. Published by Oxford University Press.