Middle ear cholesteatoma is characterized by the hyperproliferation of keratinocytes. In recent decades, N-methyladenosine (mA) modification has been shown to play an essential role in the pathogenesis of many proliferative diseases. However, neither the mA modification profile nor its potential role in the pathogenesis of middle ear cholesteatoma has currently been investigated. Therefore, this study aimed to explore mA modification patterns in middle ear cholesteatoma. An mA mRNA epitranscriptomic microarray analysis was performed to analyze mA modification patterns in middle ear cholesteatoma tissue ( = 5) and normal post-auricular skin samples ( = 5). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to predict the potential biological functions and signaling pathways underlying the pathogenesis of middle ear cholesteatoma. Subsequently, mA modification levels were verified by methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) in middle ear cholesteatoma tissue and normal skin samples, respectively. A total of 6,865 distinctive mA-modified mRNAs were identified, including 4,620 hypermethylated and 2,245 hypomethylated mRNAs, as well as 9,162 differentially expressed mRNAs, including 4,891 upregulated and 4,271 downregulated mRNAs, in the middle ear cholesteatoma group relative to the normal skin group. An association analysis between methylation and gene expression demonstrated that expression of 1,926 hypermethylated mRNAs was upregulated, while expression of 2,187 hypomethylated mRNAs and 38 hypermethylated mRNAs was downregulated. Moreover, GO analysis suggested that differentially methylated mRNAs might influence cellular processes and biological behaviors, such as cell differentiation, biosynthetic processes, regulation of molecular functions, and keratinization. KEGG pathway analysis demonstrated that the hypermethylated transcripts were involved in 26 pathways, including the Hippo signaling pathway, the p53 signaling pathway, and the inflammatory mediator regulation of transient receptor potential (TRP) channels, while the hypomethylated transcripts were involved in 13 pathways, including bacterial invasion of epithelial cells, steroid biosynthesis, and the Hippo signaling pathway. Our study presents mA modification patterns in middle ear cholesteatoma, which may exert regulatory roles in middle ear cholesteatoma. The present study provides directions for mRNA mA modification-based research on the epigenetic etiology and pathogenesis of middle ear cholesteatoma.