Toxin-antitoxin systems (TASs) have attracted much attention due to their important physiological functions. These small genetic factors have been widely studied mostly in commensalEscherichia colistrains, whereas the role of TASs in the probioticE. coliNissle 1917 (EcN) is still elusive. Here, the physiological role of chromosomally encoded type II TASs inEcN was examined. We showed that gene pairECOLIN_00240-ECOLIN_00245andECOLIN_08365-ECOLIN_08370were two functional TASs encoding CcdAB and HipAB, respectively. The homologs of CcdAB and HipAB were more conserved inE. colispecies belonging to pathogenic groups, suggesting their important roles inEcN. CRISPRi-mediated repression ofccdABandhipABsignificantly reduced the biofilm formation ofEcN in the stationary phase. Moreover,ccdABandhipABwere shown to be responsible for the persister formation inEcN. Biofilm and persister formation ofEcN controlled by theccdABandhipABwere associated with the expression of genes involved in DNA synthesis, SOS response, and stringent response. Besides, CRISPRi was proposed to be an efficient tool in annotating multiple TASs simultaneously. Collectively, our results advance knowledge and understanding of the role of TASs inEcN, which will enhance the utility ofEcN in probiotic therapy.