Toxin-antitoxin (TA) loci encode two-component genetic elements: a stable “toxin” protein and an unstable “antitoxin” (a protein or a regulatory RNA) that neutralizes the toxin action. A lot of researches have been done on the cellular targets of toxins, the molecular structures and biological functions, and the mechanisms of antitoxin actions. The studies not only discovered a multitude of physiology roles of TA systems, but also led to a number of applications for various purposes. Currently, five TA systems have been discovered in countless bacteria and archaea. The antitoxins in type Ⅰ systems are regulatory RNAs which can employ several different modes to counteract toxin proteins and their special RNA regulatory strategies have made type Ⅰ TA systems become a focus of TA researches. Within this review, we will summarize our current knowledge on type Ⅰ TA system and look into the future of their potential applications.