The sRNA is an important regulatory molecule that plays a key role in virulence and environmental adaptive responses in bacteria. To explore the role of sRNA rli34 in virulence of Listeria monocytogenes (L. monocytogenes), here, the molecular features of trans-encoded sRNA rli34 and its intracellular and extracellular expression profiles were characterized. Furthermore, the effects of rli34 gene deletion on virulence were investigated. Meanwhile, target genes regulated by rli34 were predicted and analyzed and rli34-mRNA interactions were validated using a bacterial dual plasmid reporter system based on E. coli. The results showed that rli34 was trans-encoded sRNA and highly conserved in L. monocytogenes, and its intracellular expression level was increased by 10.94-fold as compared to extracellular condition. Moreover, compared with LM EGD-e and LM-Δrli34-rli34, the adhesion, invasion, intracellular survival and cell inhibition of LM-Δrli34 on RAW264.7 cells were significantly declined (P<0.05). Furthermore, there were significant differences in bacterial loads in both liver and spleen (P<0.01), and the virulence of LM-Δrli34 was significantly dampened. Bioinformatics analysis revealed that rli34 could complementarily pair with 5'-UTR (-279 to -270) bases of wall teichoic acids (WTAs) glycosyltransferase (gtcA) mRNA, whereby it promoted the expression of gtcA gene. These findings suggested that this novel trans-encoded sRNA rli34 may facilitate the glycosylation of cell WTAs by targeting gtcA mRNA, thereby precisely modulating the virulence of L. monocytogenes. This study provided new insights into the mechanisms of trans-encoded sRNAs in virulence modulation in L. monocytogenes.