Ecological condition of the regulating and storing lakes along inter-basin water diversion project directly determines whether the project can be carried out smoothly. Based on the survey data in April and September 2018, macroinvertebrate species composition, community structure and its relationships with environmental factors were studied in the Gaoyou Lake. A total of 42 species, belonging to 3 phylum, 7 class and 22 families, were identified during the investigation period. Microchironomus tener, Limnodrilus hoffmeisteri, Nephtys sp., Branchiura sowerbyi, Teneridrilus mastix were the dominant taxa, with relative abundance were 24.0%, 17.7%, 8.1%, 7.2% and 6.5%, respectively. The dominant species compositions of the two surveys were seriously different. Macroinvertebrate density, species richness, Shannon index were all significantly higher in April than in September; non-matric multi-dimensional scaling (NMDS) and multi-response permutation procedures (MRPP) analyses confirmed the difference of macroinvertebrate community structure at community level. Indicator species analysis showed that Microchironomus tener, Limnodrilus hoffmeisteri and Aulodrilus pectinatus were the key taxa that caused community differences. Distance-based redundancy analysis (dbRDA) showed that macroinvertebrate communities in the Gaoyou Lake were mainly affected by Si(OH)₄ and total phosphorus in April, and water depth and chlorophyll a in September.