N6,2′-O-dimethyladenosine (m6Am) is a prevalent modification frequently found at the 5′ cap-adjacent adenosine of messenger RNAs (mRNAs) and small nuclear RNAs (snRNAs) and the internal adenosine of snRNAs. This dynamic and reversible modification is under the regulation of methyltransferases phosphorylated CTD interacting factor 1 and methyltransferase-like protein 4, along with the demethylase fat mass and obesity-associated protein. m6Am RNA modification plays a crucial role in the regulation of pre-mRNA splicing, mRNA stability, and translation, thereby influencing gene expression. In recent years, there has been growing interest in exploring the functions of m6Am and its relevance to human diseases. In this review, we provide a comprehensive overview of the current knowledge concerning m6Am, with a focus on m6Am-modifying enzymes, sequencing approaches for its detection, and its impacts on pre-mRNA splicing, mRNA stability, and translation regulation. Furthermore, we highlight the roles of m6Am in the context of obesity, viral infections, and cancers, unravelling its underlying regulatory mechanisms.