Chemical analysis, PCR-DGGE and DNA sequencing methods were used to investigate effects of bacterial manure varying in application rate and applied at diffentent time on soil physicochemical properties and microbial community diversity in the rhizosphere of highland barley in Tibet. Results show that application of bacterial manure significantly increased total N, total P, total K, organic matter, alkalytic N, available P and readily available K in the soil, for instance, by 13.32%, 28.42%, 16.20%, 9.81%, 21.36%, 39.35% and 30.48% respectively over that in CK when 750 ml hm^-2 of bacterial manure was applied before sowing; and by 7.25%, 29.35%, 18.04%, 12.86%, 15.90%, 43.27% and 53.99%, respectively, when 2 250 ml hm^-2 of bacterial manure was applied at the jointing stage of the crop. DGGE analysis demonstrates that DGGE atlases of microorganisms in the soils the same in application method, were all quite similar regardless of application rate. Cluster analysis with UPGMA sorted the DGGE atlases into two clusters. Shannon indices suggest that soil microbial diversity in soils applied bacterial manure increased first and decreased gradually afterwards. Application of 750 ml hm^-2 of bacterial manure prior to sowing brought about the highest soil microbial diversity, while application of 2 250 ml hm^-2 at the jointing stage did. Moreover, the former was higher than the latter in nutrient releasing and Shannon index as well. DNA sequencing shows that microbial populations were widely distributed and dominated with bacteria of Actinobacteria class, and a few uncultured strains in the soils applied with bacterial manure, regardless of application rate. Canonical correspondence analysis (CCA) reveals that the distribution of DGGE fingerprints was strongly related to physical and chemical properties of the soil. AN (Alkalytic N), TP and TN were the main environmental factors influencing structure of the microbial community. All the findings demonstrate that application of bacterial manure significantly improves physicochemical properties of the soil and microbial community diversity of the rhizosphere soil of highland barley.