Three Gorges Reservoir was impounded to 175 m for the first time in October 2010, of which the backwater area arrived in Chongqing, and the upstream water retention time extended further and the reaches changed to transitional waters. The heterogenrity of protozoan communities was studied in the main stem of Three Gorges Reservoir during the period of Three Gorges Reservoir normal operation (October 2010-June 2011). The spatial variations in community structure and species distribution were observed during reservoir drainage and storage. A total of 99 protozoan species were identified. Of these taxa, ciliates represented an increasing tendency after impoundment. The dominant species differed in operating stages, with the indicator species generally evolved from tintinnids (impounding period), ciliates (high water level period) to testate amoebae (low water level period). The dominant species varied in different reservoir areas, while the indicator species testate amoebae (transition zone) were changed into ciliates (lake zone) from upstream to downstream.Detrended correspondence analysis (DCA) and multi-response permutation procedure (MRPP) analysis showed that protozoan community structures and species distributions showed a clear spatio-temporal heterogeneity in Three Gorges Reservoir. The temporal heterogeneity peaked during impounding stage (P < 0.05). The statistical analysis showed that the spatio-temporal variations in community pattern and species distribution were significantly correlated to environmental variables, especially transparency, temperature, conductivity and chlorophyll a. The average density and biomall of protozoa were 952.19 ind./L and 8.14 g/L, respectively. The standing crops of protozoa in upstream were higher than that in downstream during impounding stage, but lower than that during low water level period. The mean values of Marglef's and Shannon-Weiner diversity indices were 3.78 and 2.18, respectively, with minimum value in January and maximum in June. The abundance of protozoa in transition zone was high after 175 m impoundment. It may be due to large amount of organic matter brought by water flow during the first impounding stage. Reservoir operation pattern of "drainage in winter and storage in summer" "blurs" seasonal variation of the river. Protozoan species composition and standing crops are mainly affected by reservoir hydrodynamic. Physic-chemical factors and hydrological characteristics showed different spatio-temporal variations, and resulted in the heterogeneity in community pattern and species distribution of protozoa on both temporal and spatial scales.