Tetragenococcus halophilus is a moderately halophilic lactic acid bacterium widely used in high-salt food fermentation because of its coping ability under various stress conditions. Bacterial toxin-antitoxin (TA) modules are widely distributed and play important roles in stress response, but those specific for genus Tetragenococcus have never been explored. Here, a bona fide TA module named DinJ(1)-YafQ(1)(tha) was characterized in T. halophilus. The toxin protein YafQ(1)(tha) acts as a ribonuclease, and its overexpression severely inhibits Escherichia coli growth. These toxic effects can be eliminated by introducing DinJ(1)(tha), indicating that YafQ(1)(tha) activity is blocked by the formed DinJ(1)-YafQ(1)(tha) complex. In vivo and in vitro assays showed that DinJ(1)(tha) alone or DinJ(1)-YafQ(1)(tha) complex can repress the transcription of dinJ(1)-yafQ(1)(tha) operon by binding directly to the promoter sequence. In addition, dinJ(1)-yafQ(1)(tha) is involved in plasmid maintenance and stress response, and its transcriptional level is regulated by various stresses. These findings reveal the possible roles of DinJ(1)-YafQ(1)(tha) system in the stress adaptation processes of T. halophilus during fermentation. A single antitoxin DinJ(2)(tha) without a cognate toxin protein was also found. Its sequence shows low similarity to that of DinJ(1)(tha), indicating that this antitoxin may have evolved from a different ancestor. Moreover, DinJ(2)(tha) can cross-interact with noncognate toxin YafQ(1)(tha) and cross-regulate with dinJ(1)-yafQ(1)(tha) operon. In summary, DinJ-YafQ(tha) characterization may be helpful in investigating the key roles of TA systems in T. halophilus and serves as a foundation for further research.