DNA methylation, a well-known epigenetic modification in mammalian genomes, is important for development and health. Dysregulation of DNA methylation can cause abnormal gene regulation, leading to anomalous development and diseases. Until recently, the ability to understand the functions and dynamics of DNA methylation was limited by the availability of technologies for comprehensively characterizing methylation on a genome-wide scale. Rapid advances in high-throughput approaches (particularly next-generation sequencing), coupled with molecular techniques, have enabled unbiased genome-wide profiling of DNA modifications at single-base resolution and helped to elucidate their impact on gene regulation. Here, we discuss the development of genomic approaches to decipher the global methylome at single-base resolution, the challenges faced, and the emerging new insights. Our ability to decipher this important epigenetic modification and how it impacts gene expression will provide a framework for understanding numerous disease mechanisms, and suggest means to treat or prevent them in the future.