The root microbiome plays a key role that can influence host plant growth and abiotic stress. While there has been extensive characterization of community structure, spatial compartmentalization, and the impact of drought stresses on the root microbiome in rice and other plants, there is relatively little known about the differences in root microbiome among rice ecotypes in natural upland and lowland fields. Herein, we used two rice ecotypes, upland and irrigated ecotype rice (two Indica and two Japonica genotypes), as a model to explore the responses of the root microbiome under different environmental conditions. We aimed to identify environment-induced adaptation in the root bacterial and fungal composition of rice ecotypes by high-throughput sequencing. Rice from lowland field or upland had significantly altered overall bacterial and fungal community compositions of the two ecotypes, with diversity of both ecotypes greatly decreased from lowland field to upland. The overall response of the root microbiome to upland conditions was taxonomically driven by the enrichment of family Enterobacteriaceae and genera Serratia, and phylum Ascomycota. Interestingly, rice ecotypes specifically enriched root microbes when they were transferred from their original environment, such as the enrichment of class Thermoleophilia and phylum Actinobacteria when the irrigated ecotype rice was moved from lowland to upland field. These results revealed that different environmental conditions and rice ecotypes resulted in a restructuring of root microbiome communities, and suggested the possibility that components responsible for the beneficial attributes in the altered root microbiome might contribute to the adaptation of different ecotypes in natural fields.