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As a standard strain of American strains library widely used in scientific research, Escherichia coli ATCC 8739 was used in microarray analysis experiment to study the antimicrobial activity of cinnamaldehyde (CMA). The reverse transcription (RT)-PCR and western blot experiments were used to verify the results of microarray analysis. The microarray analysis showed that 126 genes expressed differentially, where the number of upregulated genes was 83 (65.9%) and downregulated genes was 43 (34.1%). These differentially expressed genes were divided into 16 classes including hypothetical protein (19.8%), metabolism of coenzymes, prosthetic groups (11.9%) and so on. The results of RT-PCR and western blot experiments coincide with the microarray analysis, so the microarray analysis study was effective. These results indicated that flagellin and cell membrane may be the preliminary antibacterial mechanism of CMA. CMA could affect the synthesis and aggregation of cytoplasmic protein and interfere with ATP metabolism. These effects of CMA could lead to bacterial metabolic disorder and autolysis.