ABSTRACT: BACKGROUND: GeoChip 3.0, a microbial functional gene array, containing ~28,000 oligonucleotide probes and targeting ~57,000 sequences from 292 functional gene families, provided a powerful tool for researching microbial community structure in natural environments. The alpine meadow is a dominant plant community in the Qinghai-Tibetan plateau, hence it is important to profile the unique geographical flora and assess the response of the microbial communities to environmental variables. In this study, Geochip 3.0 was employed to understand the microbial functional gene diversity and structure, and metabolic potential and the major environmental factors in shaping microbial communities structure of alpine meadow soil in Qinghai-Tibetan Plateau. RESULTS: A total of 6143 microbial functional genes involved in carbon degradation, carbon fixation, methane oxidation and production, nitrogen cycling, phosphorus utilization, sulphur cycling, organic remediation, metal resistance, energy process and other category were detected in six soil samples and high diversity was observed. Interestingly, most of the detected genes associated with carbon degradation were derived from cultivated organisms. To identify major environmental factors in shaping microbial communities, Mantel test and CCA Statistical analyses were performed. The results indicated that altitude, C/N, pH and soil organic carbon were significantly (P < 0.05) correlated with the microbial functional structure and a total of 80.97% of the variation was significantly explained by altitude, C/N and pH. The C/N contributed 38.2% to microbial functional gene variation, which is in accordance with the hierarchical clustering of overall microbial functional genes. CONCLUSIONS: High overall functional genes and phylogenetic diversity of the alpine meadow soil microbial communities existed in the Qinghai-Tibetan Plateau. Most of the genes involved in carbon degradation were derived from characterized microbial groups. Microbial composition and structures variation were significantly impacted by local environmental conditions, and soil C/N is the most important factor to impact the microbial structure in alpine meadow in Qinghai-Tibetan plateau.