Cucumber (Cucumis sativus) is one of the most widely used vegetable in the world, and different pesticides have been extensively used for controlling the insects and disease pathogens of this plant. However, little is known about how the pesticides affect the microbial community in cucumber phyllosphere. This study was the first attempt to assess the impact of pyrethroid insecticide cyperemethrin on the microbial communities of cucumber phyllosphere using biochemical and genetic approaches. Phospholipid fatty acid (PLFA) assay indicated that cyperemethrin insecticide treatment led to a significant increase in both total and bacterial biomass and a decrease in fungal biomass and the ratio of Gram-positive (GP) bacteria to Gram-negative (GN) bacteria within the cucumber phyllosphere. Principal-component analyses (PCA) suggested that the number of unsaturated and cyclopropane PLFAs (16:1ω9t,18:1ω7, cy17:0, cy19:0) increased with the insecticide treatment, whereas the saturated PLFA i16:0, i17:0 decreased. The increase of GN bacteria implied that the cypermethrin insecticide might be a nutrient for the growth of these phyllosphere microbes. Terminal restriction fragment length polymorphism (T-RFLP) reinforced the PLFA results. A significant change of bacterial community structure was observed in the separate dendrogram cluster between control and treated samples with the cucumber phyllosphere following cypermethrin insecticide treatment. Moreover, the increased terminal restriction fragments (T-RFs) (58, 62, 89, 99, 119, 195, 239,311,340, and 473 bp) indicated that some bacteria might play a significant role in the insecticide degradation within the cucumber phylosphere, whereas the disappeared T-RFs (44, 51, 96, 223, 306, and 338 bp) implied that some other bacteria might potentially serve as microbial indicator of cyperemethrin insecticide exposure.