The effects of transgenic plant on ecological and food biosafety have attracted considerable attention recently due to the exploding number of commercialization genetic modified plants. Here, we examined the changes of bacterial and fungal community compositions in the phyllosphere of Cry1Ac Bacillus thuringiensis (Bt) cotton SGK321 by molecular fingerprint method at four major growth stages. The phyllospheric bacteria diversity of SGK321 was decreased at budding stage only, indicated by Shannon index. A slight increase of diversity and abundance of fungal community could be detected at different sampling stage except the seeding stage. Fungal community showed a tendency to be affiliated with the expression of Cry1Ac protein which was detected by enzyme-linked immunosorbent assay (ELISA) method at both the seeding and budding stages. Results suggested that, compared to bacteria, fungi in phyllosphere of transgenic cotton SGK321 might be more susceptible to Cry1Ac protein, which includes Cochliobolus sp., Setosphaeria sp., Blakeslea trispora, Cryptococcus neoformans and Cryptococcus sp. Furthermore, the seasonal and temporal parameters during the growth stages were proved to be the most important factor relating with the shift of phyllospheric microorganism communities.