Non-coding RNAs (ncRNAs) have gained increasing attention as their diverse roles in virulence and environmental stress in Listeria monocytogenes have become clearer. The ncRNA rliB is an atypical member of the CRISPR family, conserved at the same genomic locus in all analyzed L. monocytogenes genomes and also in other Listeria species. In this study, rliB defective mutants (Lm3-22-ΔrliB) were constructed by homologous recombination. The growth cycle of Lm3-22-ΔrliB mutants was slower than that of wild-type Lm3-22. The sensitivity of Lm3-22-ΔrliB to the Listeria phage vB-LmoM-SH3-3 was significantly increased, and the efficiency of plaque formation was enhanced by 128 fold. Compared with wild type, the adhesion and invasion of Lm3-22-ΔrliB decreased significantly (9.3% and 1.33%, respectively). After 4 hours of infection, the proliferation of Lm3-22-ΔrliB in RAW264.7 cells also decreased significantly. Transcription level of invasion-related surface proteins showed that the internalin genes lmo0610 and lm0514, and the peptidoglycan binding protein gene lmo1799 in Lm3-22-ΔrliB were significantly increased. In addition, after interaction with phage, the transcription levels of inlA, lmo0610, lmo1799, lmo2085, and lmo0514 in Lm3-22-ΔrliB cells were significantly upregulated, while inlB was downregulated, compared with Lm3-22 control group with phage treatment. Therefore, rliB deletion effectively regulated the interaction between Listeria and phage, weaken its invasion ability, and provided a new theoretical basis for biocontrol of phage.