Adenylate cyclase Ⅲ (AC3) is an important component of odorant perception signaling in the olfactory system. The thinness of main olfactory epithelium (MOE) become thin with ageing, and the gene expression profile alters after AC3 deletion. DNA methylation plays a key role in animal development and regulation of gene expression. In the present study, whether the DNA methylation level of gene promoter, as well as their associations with the expression of the genes in MOE will be altered after AC3 deletion, was investigated by using methylated DNA immunoprecipitation chip (MeDIP-chip), methylation-specific PCR (MSP) and real-time fluorescence quantification PCR. The data showed that the DNA methylation levels of promoters of 1 978 genes were altered in AC3-deficient mice, accounting for 9% of the total number of genes. Of which 727 genes with their promoter’s DNA methylation levels were elevated, 1 251 genes with their promoter’s methylation levels were lowered. The functions of these genes are mainly involved with olfactory receptor, neurodevelopmental, cAMP signaling pathway, ATP-binding, calcium regulation, acetylation modification, and transcription factors. It was further confirmed by MSP that methylation levels of promoter of the olfactory receptor genes Olfr1153, Olfr231, Olfr378, Olfr651 and Olfr691 were increased, whereas methylation level of the promoters of Cngb1, Pde4a and Olfr1394 were decreased. In line with MSP results, qRT-PCR data showed that the expression levels of Cngb1, Hcn4, Olfm1, Olfr1394, Olfr1153, Olfr231, Olfr378 and Olfr691 were significantly decreased, whereas the expression levels of Pde4a and Olfr651 were significantly increased. In conclusion, the methylation levels of promoters of olfactory receptor genes, neurodevelopmental related genes and cAMP signaling pathways in MOE are modified significantly after AC3 deletion, which affected the transduction of signal pathways such as nucleotide excision and repair, DNA replication and mismatch repair, thus comprehensively regulating the number and level of gene expression in MOE of mice.