Restriction-modification systems (RMS) are ubiquitous in prokaryotes and serve as primitive immune-like mechanisms that safeguard microbial genomes against foreign genetic elements. Beyond their well-known role in sequence-specific defense, RMS also contribute significantly to genomic stability, drive evolutionary processes, and mitigate the deleterious effects of mutations. This review provides a comprehensive synthesis of current insights into RMS, emphasizing their structural and functional diversity, ecological and evolutionary roles, and expanding applications in biotechnology. By integrating recent advances with an analysis of persisting challenges, we highlight the critical contributions of RMS to both fundamental microbiology and practical applications in biomedicine and industrial biotechnology. Furthermore, we discuss emerging research directions in RMS, particularly in light of novel technologies and the increasing importance of microbial genetics in addressing global health and environmental issues.